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ADME PK

Reactive metabolite assessment (glutathione trapping)

  • Reactive metabolite formation is thought to be one of the primary causes of idiosyncratic adverse drug reactions, often associated with drug-induced skin, liver and hematopoietic toxicities.
  • Reactive metabolites, formed via drug metabolism in the body, are electrophilic species which can bind covalently to macromolecules such as proteins and DNA, affecting their function and potentially leading to toxicity.
  • To minimize the risk of later stage failure - which is of considerable financial burden to the Pharmaceutical Industry - screening for reactive metabolite formation at an early stage in lead optimization is now common practice1.
  • Chemical trapping agents, such as reduced glutathione (GSH), can form stable adducts with many reactive species. Trapping agents, incubated with liver microsomes, are now routinely used in the identification of reactive metabolites.
  • Cyprotex now offer glutathione trapping studies using human liver microsomes and Q-TOF high resolution accurate mass instrumentation.
  • By using high resolution accurate mass spectrometry, it improves detection of the conjugates and allows superior structural characterisation. The process utilises MSE data acquisition, mass detect filtering and post acquisition data mining.
Screening and structural characterization of reactive metabolites, as one of the major efforts to reduce attrition in drug development, has increasingly become an integral part of the ADMET-guided lead optimization process in drug discovery.

2 Yan Z, Maher N, Torres R, Caldwell GW and Huebert N (2005) Rapid Commun Mass Spectrom 19; 3322-3330

Protocol

Reactive metabolite formation by glutathione trapping

Assay Design Test article incubated with human liver microsomes and glutathione in the presence and absence of NADPH
Test Article Concentration 50 µM
Microsomal Concentration 1 mg/mL
Glutathione Concentration 1 mM
Quality Controls Minus NADPH (negative control)
Ticlopidine (positive control)
Test Article Requirements 100 µL of 10 mM DMSO solution or equivalent amount of solid compound.
Analysis Method High resolution accurate mass Q-TOF
Data Delivery Summary report including:
  • LC-MS chromatograms of the parent and reactive metabolites, along with spectra with and without fragmentation
  • Table including mass, name of proposed metabolite and formula, m/z found, mass error, retention time, absolute area and area percentage
Structural elucidation (optional)

Comprehensive report (optional)

Data

Data for reactive metabolite formation (glutathione trapping)

 
Figure 1
Representative XIC chromatogram of ticlopidine following incubation with human liver microsomes and glutathione in the absence and presence of NADPH.

In the presence of NADPH, reactive metabolite formation is evident. M1 represents a GSH adduct + reduction; M2 and M3 represent GSH adducts + hydration.
 
Figure 2
MSMS spectrum for ticlopidine following incubation with human liver microsomes, glutathione and NADPH.

The spectrum illustrates confirmation of reactive metabolite M2 through neutral losses for GSH and hydration.
NameFormulaMass Differencem/zMass error (ppm)IdentifierRT (min)Neutral loss
Parent C14H14ClNS - 264.0618 1.7 - 3.23 -
GSH + reduction C24H31ClN4O6S2 +307.0830 571.1443 -1.5 M1 2.67 307
GSH + hydration C24H31ClN4O7S2 +323.0790 587.1404 0.6 M2 2.45 75, 129, 307
GSH + hydration C24H31ClN4O7S2 +323.0785 587.1398 -0.5 M3 2.52 129, 307

Table 1

Table illustrating representative data for ticlopidine following incubation with human liver microsomes and glutathione in the presence of NADPH.

References

1 Evans DC et al. (2004) Drug−protein adducts:  An industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem Res Toxicol 17(1); 3-16

2 Yan Z et al. (2005) Rapid detection and characterization of minor reactive metabolites using stable-isotope trapping in combination with tandem mass spectrometry. Rapid Commun Mass Spectrom 19(22); 3322-3330

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