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