Glutathione (GSH) 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 Glutathione (GSH) Trapping Assay is based upon microsomal incubation of parent compound in the presence of an excess of reduced glutathione. Reactive metabolites formed during the incubation are trapped by GSH and are detected using high resolution mass spectrometry. This assay is particularly appropriate for molecules with a potential to form soft-electrophiles, such as Michael-acceptor, as reactive metabolites (e.g clozapine).
|Compound requirements||5 mg or 50 µL of 50 mM stock solution (in DMSO)|
|Test compound concentration||50 µM|
|Time points||0 and 90 min|
|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 GSH adducts are detected using manual data interrogation of the diagnostic product ion at m/z 272.0888 in the resultant negative-ion AIF (all-ion fragmentation) spectrum. This is highly selective and provides wide coverage for detection of unexpected GSH adducts.
We will report:
- Assessment of metabolic turnover of positive control compound
- Detected GSH adducts with proposed molecular formula
- Assessment of metabolic vs. chemical formation (using +/- NADPH controls)
- Extracted mass chromatograms of parent and GSH adducts for test compound and positive control
|ID||Proposed transformation||Retention time (min)||Observation ion [M-H]–||Molecular Formula|
|CLZ-7||P + (GSH – 2H) + O||3.57||646.1866||C28H34N7O7ClS|
|CLZ-8||P + (GSH – 2H)||3.69||630.1914||C28H34N7O6ClS|