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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
Positive control Clozapine
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.

Example of diagnostic m/z 272 product ion spectra

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

Extracted mass chromatogram (MS2 negative) for m/z 272 fragment

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

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