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Cyanide Trapping Assay

Drug metabolism can generate chemically reactive metabolites which have the potential to react with cellular components, such as DNA and proteins, altering their function and risk inducing idiosyncratic toxicity. This is a major issue for patient safety and has led to drug withdrawals and black box warnings.

Early assessment of candidate compounds for reactive metabolite formation allows the identification and the mitigation of these risks. Sygnature offers the Glutathione (GSH) Trapping assay and the Cyanide Trapping assay for the evaluation of reactive metabolites formation.

Our fully validated Cyanide Trapping Assay is based upon microsomal incubation of parent compound in the presence of a cyanide source (KCN). Reactive metabolites formed during the incubation are trapped using a mixture of isotopically-labelled and unlabelled potassium cyanide and are detected using accurate-mass high-resolution mass spectrometry. The distinctive isotopic pattern increases confidence in the detection of low-level metabolites and helps reduce false positives. This assay is particularly appropriate for molecules with a potential to form hard-electrophiles, such as iminium ions generated by tertiary amines, as reactive metabolites (e.g. nicotine).

Compound requirements 1 mg or 50 µL of 10 mM stock solution (in DMSO)
Test compound concentration 10 or 50 µM
Time points 0 and 90 min
Positive control Clozapine and nicotine
Cyanide source KCN/K13C15N
Analysis Method ThermoFisher Q Exactive Focus mass spectrometer in full scan and AIF (all ion fragmentation) data acquisition with positive/negative polarity switching

The trapped cyanide adducts are detected using their distinctive isotopic pattern by accurate-mass high-resolution mass spectrometry using manual and software-assisted (Thermo Scientific Compound Discoverer) data interrogation.

Example of CN-adduct mass spectra

We will report:

  • Assessment of metabolic turnover of positive control compound
  • Detected cyanide adducts with proposed molecular formula
  • Assessment of metabolic vs. chemical formation (using +/- NADPH controls)
  • Extracted mass chromatograms of parent and cyanide adducts for test compound and positive control

1. Extracted mass chromatograms of parent and CN adducts of Clozapine

2. Extracted mass chromatograms of observed CN adducts of clozapine

ID Proposed transformation Retention time (min) Observation ion [M-H] Molecular Formula
P Parent 3.11 327.1356 C18H19ClN4
CLZ – CN1 -Cl +OH (-H +CN) 2.54 334.1652 C19H19N5O
CLZ – CN2 +O (-H +CN) 2.71 368.1261 C19H18ClN5O

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