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| 1mg |
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| Targets |
N-Acetyl sulfapyridine-d4 is a deuterium-labeled analytical standard and does not directly target biological receptors. The non-labeled parent compound, N-Acetyl sulfapyridine, is the major active metabolite of sulfasalazine. Sulfasalazine is a prodrug that is cleaved by intestinal bacteria into 5-aminosalicylic acid (5-ASA) and sulfapyridine. Sulfapyridine is absorbed and extensively metabolized, primarily by acetylation via N-acetyltransferase 2 (NAT2), to N-Acetyl sulfapyridine. The therapeutic effect of sulfasalazine in IBD is primarily attributed to 5-ASA, while sulfapyridine and its metabolites are responsible for most of the adverse effects. N-Acetyl sulfapyridine has been implicated in the adverse effects of sulfasalazine, including nausea, headache, and rash. The deuterated version is used solely as an analytical standard.
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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].
As a deuterium-labeled internal standard, N-Acetyl sulfapyridine-d4 itself does not possess intrinsic in vitro biological activity. It is used exclusively for analytical quantification purposes. The non-labeled N-Acetyl sulfapyridine is a metabolite of sulfasalazine and has been associated with adverse effects. In vitro, sulfasalazine and its metabolite sulfapyridine have been shown to inhibit the proliferation of lymphocytes and the production of pro-inflammatory cytokines. N-Acetyl sulfapyridine has lower activity than sulfapyridine. The compound can be detected in human serum and urine, and its concentration is used as a marker of NAT2 acetylation status (slow vs. fast acetylator phenotype). The deuterated version is used as an internal standard to accurately quantify N-Acetyl sulfapyridine in biological samples, such as serum, plasma, and urine, for pharmacokinetic and pharmacogenetic studies. |
| ln Vivo |
N-Acetyl sulfapyridine-d4 does not exhibit in vivo biological activity because it is an analytical standard. The non-labeled N-Acetyl sulfapyridine is the major active metabolite of sulfasalazine and is associated with the adverse effects of the drug. After oral administration of sulfasalazine, it is cleaved by intestinal bacteria to 5-ASA and sulfapyridine. Sulfapyridine is absorbed and metabolized by N-acetyltransferase 2 (NAT2) to N-Acetyl sulfapyridine. The rate of acetylation is genetically determined; individuals with slow acetylator phenotype (NAT2*5, *6, *7) have higher plasma concentrations of sulfapyridine and are at increased risk of adverse effects. The plasma concentration of N-Acetyl sulfapyridine is often measured in clinical studies to assess acetylation status and to monitor drug therapy. As an internal standard, the deuterated form is used to accurately quantify N-Acetyl sulfapyridine in biological samples.
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| Enzyme Assay |
A typical non-cellular protocol for using N-Acetyl sulfapyridine-d4 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 methanol or DMSO. For serum or plasma samples, 50 uL of serum 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 N-Acetyl sulfapyridine and the deuterated 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 N-Acetyl sulfapyridine-d4 as an internal standard involves the quantification of the metabolite in cultured hepatocytes. Primary human hepatocytes or HepG2 cells are seeded in 6-well plates at a density of 1×10⁶ cells/well and cultured in appropriate medium for 24 hours. After treatment with sulfapyridine (non-labeled, 1-100 uM) for various time periods, the culture medium is collected. 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 sonication (3×10 seconds on ice). Proteins are precipitated by adding 800 uL of acetonitrile containing 0.1% formic acid. After centrifugation, the supernatant is analyzed by LC-MS/MS. The concentration of N-Acetyl sulfapyridine in the culture medium and cell lysate is quantified using a calibration curve prepared with the same internal standard. The internal standard corrects for extraction efficiency and matrix effects.
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| Animal Protocol |
A typical in vivo animal protocol for using N-Acetyl sulfapyridine-d4 as an internal standard involves the quantification of sulfasalazine metabolites in plasma and urine. Male Sprague-Dawley rats (200-250 g) are administered sulfasalazine (non-labeled) via oral gavage at a dose of 100-200 mg/kg. Blood samples (200 uL) are collected via the tail vein into EDTA-coated tubes at various time points (0, 1, 2, 4, 6, 8, 12, 24 hours) post-dose. Plasma is separated by centrifugation (2,000 g, 10 minutes, 4degC). For extraction, 50 uL of plasma is mixed with 10 uL of N-Acetyl sulfapyridine-d4 internal standard solution (1 ug/mL) and 150 uL of acetonitrile. After centrifugation, the supernatant is analyzed by LC-MS/MS. The concentrations of sulfapyridine and N-Acetyl sulfapyridine are quantified using calibration curves prepared with the same internal standard. The internal standard corrects for variations in sample preparation and matrix effects.
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| ADME/Pharmacokinetics |
As an analytical internal standard, N-Acetyl sulfapyridine-d4 is not characterized by typical pharmacokinetic parameters. The non-labeled N-Acetyl sulfapyridine is the major metabolite of sulfasalazine. After oral administration of sulfasalazine, sulfapyridine is released and then acetylated to N-Acetyl sulfapyridine. The pharmacokinetics of sulfapyridine and its acetylated metabolite are dependent on the N-acetyltransferase 2 (NAT2) genotype. In slow acetylators, the plasma half-life of sulfapyridine is longer (approximately 10-20 hours) compared to fast acetylators (5-10 hours). The concentration of N-Acetyl sulfapyridine is higher in fast acetylators. The deuterated internal standard is used to accurately quantify N-Acetyl sulfapyridine in biological samples for pharmacokinetic and pharmacogenetic studies.
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| Toxicity/Toxicokinetics |
Toxicity data specific to N-Acetyl sulfapyridine-d4 are not available. The non-labeled N-Acetyl sulfapyridine is a metabolite of sulfasalazine and is associated with adverse effects such as nausea, headache, rash, and, rarely, more serious reactions such as Stevens-Johnson syndrome and agranulocytosis. The risk of adverse effects is higher in slow acetylators (NAT2 slow acetylator genotype) due to the accumulation of sulfapyridine. Standard laboratory safety precautions should be followed when handling N-Acetyl sulfapyridine-d4, including the use of gloves, lab coats, and safety glasses. The compound should be stored at -20degC, protected from light and moisture. It is for research use only and should not be used in humans or animals for therapeutic or diagnostic purposes.
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| References | |
| Additional Infomation |
N-Acetyl sulfapyridine-d4 (CAS# 1189732-52-2) is a stable isotope-labeled compound with a molecular weight of 295.35. The molecular formula is C13H9D4N3O3S, and it is also known as N-Acetyl Sulfapyridine-d4 (Major) and N-(4-(N-(pyridin-2-yl)sulfamoyl)phenyl-2,3,5,6-d4)acetamide. The isotopic purity is typically greater than 98%, and the chemical purity is ≥95%. This deuterated compound is the major active metabolite of the anti-inflammatory drug sulfasalazine and is related to the adverse effects of sulfasalazine in human serum. It aids in precise quantification of sulfapyridine and its metabolites in biological matrices and supports research into drug metabolism, acetylation processes, and sulfonamide pharmacodynamics, especially in relation to slow and fast acetylator phenotypes in personalized medicine. This product is for research use only and is not approved for clinical diagnostic applications.
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| Molecular Formula |
C13H9D4N3O3S
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| Molecular Weight |
295.35
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| Exact Mass |
295.093
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| CAS # |
1189732-52-2
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| PubChem CID |
46780055
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| Appearance |
White to off-white solid powder
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| LogP |
3.644
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
20
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| Complexity |
425
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[2H]C1=C(C(=C(C(=C1NC(=O)C)[2H])[2H])S(=O)(=O)NC2=CC=CC=N2)[2H]
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| InChi Key |
CYLYVXPHAQLXFG-KDWZCNHSSA-N
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| InChi Code |
InChI=1S/C13H13N3O3S/c1-10(17)15-11-5-7-12(8-6-11)20(18,19)16-13-4-2-3-9-14-13/h2-9H,1H3,(H,14,16)(H,15,17)/i5D,6D,7D,8D
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| Chemical Name |
N-[2,3,5,6-tetradeuterio-4-(pyridin-2-ylsulfamoyl)phenyl]acetamide
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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 | 3.3858 mL | 16.9291 mL | 33.8581 mL | |
| 5 mM | 0.6772 mL | 3.3858 mL | 6.7716 mL | |
| 10 mM | 0.3386 mL | 1.6929 mL | 3.3858 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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