CAS 153466-65-0 Diclofenac-d4 - Analytical Products / Alfa Chemistry

CAT	APS153466650
CAS	153466-65-0
Structure
Synonyms	6-[(2,6-Dichlorophenyl)amino]-benzen-2,3,4,5-d4-acetic acid, Diclofenac (phenyl D4), Diclofenac D4 acid (phenyl D4)
IUPAC Name	2-[2,3,4,5-tetradeuterio-6-(2,6-dichloroanilino)phenyl]acetic acid
Molecular Weight	300.17
Molecular Formula	C142H4H7Cl2NO2
Canonical SMILES	[2H]c1c([2H])c([2H])c(Nc2c(Cl)cccc2Cl)c(CC(=O)O)c1[2H]
InChI	InChI=1S/C14H11Cl2NO2/c15-10-5-3-6-11(16)14(10)17-12-7-2-1-4-9(12)8-13(18)19/h1-7,17H,8H2,(H,18,19)/i1D,2D,4D,7D

Accurate Mass	299.0418
Format	Neat
Subcategory	Stable isotope labelled compounds, Insecticides and metabolites, Enzyme inhibitors
Type	Stable Isotope Labelled

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Case Study

Diclofenac-d4 Used for the Quantitative Imaging of Diclofenac and Its Metabolites in Mouse Tissue via Nano-DESI MSI

Sanchez, Daniela Mesa, et al. Analytica Chimica Acta 1233 (2022): 340490.

Diclofenac-d4 was employed as a stable isotopic internal standard for the quantitative analysis of diclofenac and its major metabolites in murine liver and kidney tissues using nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI). This study aimed to assess the spatial localization and concentration of diclofenac, hydroxydiclofenac, and diclofenac acyl glucuronide in biological tissues following drug administration.
Calibration standards ranging from 1 nM to 5 μM were prepared in 50/50 (v/v) acetonitrile/water with 0.1% formic acid. Diclofenac-d4 at a fixed concentration of 50 nM was spiked into each tissue sample prior to extraction, enabling accurate quantification. Tissue sections were subjected to solvent-assisted sonication at 37 °C and centrifugation, with the supernatants analyzed via LC-MS/MS and nano-DESI MSI.
The nano-DESI probe maintained a stable liquid bridge between capillaries, allowing continuous analyte transfer and ionization. Diclofenac-d4 was used to normalize variations in signal intensity and extraction efficiency during imaging, facilitating reliable quantification of endogenous and exogenous compounds.
In mass spectra, diclofenac-d4 ([M - H]⁻ at m/z 298.0348) served as a robust reference peak for detecting the corresponding native species (m/z 294.0098) and their metabolites. This application underscores the essential role of diclofenac-d4 in quantitative MSI workflows for pharmacokinetic and drug distribution studies.

Diclofenac-d4 Used as an Internal Standard for the Measurement of CYP450 Activity in HepaRG Cells

Ferron, Pierre-Jean, et al. Toxicology in Vitro 33 (2016): 136-146.

In the study of cytochrome P450 (CYP450) activities, diclofenac-d4 served as a crucial internal standard (IS) to quantify the metabolism of various substrates in HepaRG cells. The assay involved incubating HepaRG cells with a cocktail containing phenacetin (CYP1A2), diclofenac (CYP2C9), (±-)-mephenytoin (CYP2C19), and midazolam (CYP3A4) for 120 minutes at 37 °C. After incubation, the media were collected, diluted in acetonitrile, and spiked with diclofenac-d4 at 5 μM.
To assess the metabolic activity, the samples were centrifuged, followed by dilution in the mobile phase A before being analyzed using LC-MS/MS for the quantification of metabolites. Protein quantification was also performed to standardize enzyme activity, measured in pmol of substrate metabolized per mg of protein per minute.
Diclofenac-d4 facilitated accurate normalization in the LC-MS/MS analysis, ensuring the reliability of CYP450 activity measurements across different substrates and conditions. This methodology enabled the precise determination of CYP450 isoform activities, which are critical for understanding drug metabolism and interaction profiles in hepatic systems.

Diclofenac-d4 Used as an Internal Standard for the Quantification of Diclofenac in Postmortem Samples

Szpot, Paweł, Olga Wachełko, and Marcin Zawadzki. Toxics 10.8 (2022): 421.

In forensic toxicology, diclofenac-d4 was employed as an internal standard for the sensitive quantification of diclofenac in postmortem samples. This study aimed to establish a robust method for detecting diclofenac in various postmortem cases, including fetal exposure, duodenal ulcer perforation, traffic accidents, and acute renal failure. The analysis utilized liquid-liquid extraction of the samples followed by ultra-high performance liquid chromatography (UHPLC) coupled with triple quadrupole tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode.
Diclofenac-d4, an isotopically labeled internal standard, was prepared in methanol and stored at -20 °C. The use of diclofenac-d4 allowed for precise quantification by compensating for potential variations during the extraction and analysis process. Electrospray ionization was employed, and the optimal monitoring of diclofenac was achieved using m/z transitions of 297.3→214.3 and other characteristic transitions for diclofenac-d4 at m/z 301.5→220.2.
The method was successfully applied in six postmortem cases, offering a sensitive and reliable approach for the detection and quantification of diclofenac in complex biological matrices. This technique proves essential for forensic investigations, enabling accurate drug distribution analysis in postmortem toxicology.