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Metabolite Identification

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Establish the metabolic profile of a promising compound early in the drug-discovery process

Metabolic profiling of therapeutic compounds at early stages in the drug discovery process is of increasing importance. Drugs that undergo metabolism in vivo may produce pharmacologically active or chemically reactive metabolites which can produce unexpected effects or potential toxicities. The recent FDA Guidance for Industry on Safety Testing of Drug Metabolites1 highlights the relevance of in vitro metabolite profiling early in drug development, as metabolites which are unique to or disproportionate in humans may require additional toxicological studies.

In addition to an extensive panel of in vitro metabolism screening assays, Eurofins is able to perform studies to thoroughly characterize the metabolic products of a drug compound in vivo or in vitro and compare metabolic profiles across species. The key element in metabolite identification studies is the use of state-of the art mass spectrometry instrumentation for sensitive and accurate detection of metabolic products and software tools to efficiently process the complex datasets that are produced from these analyses.

Eurofins employs a Waters Xevo G2 QTOF MS system combined with Acquity UPLC separation to provide high mass accuracy and excellent sensitivity in full scan MS and MS/MS modes. The acquisition method on the Xevo collects precursor and product ion data in two distinct but parallel acquisition functions via rapid switching, so that complete metabolite identification data can be obtained from a single LC injection. If necessary, follow-up selected reaction monitoring or product ion or neutral loss scans can be performed on a triple-quadrupole MS system in order to obtain corroborating data for putative metabolites.

By partnering with Eurofins, you'll be able to better understand your drug candidate's metabolism and stability and obtain a more comprehensive assessment of that compound's metabolic profile.

Flexible options to meet your research needs

Metabolism data on test compounds can be obtained through a choice of experiments, depending on the level of data depth and interpretation required:

Option Incubation Deliverables
Enhanced Metabolic Stability
(Metabolic soft-spot analysis)
Liver microsomes, S9, hepatocytes
✓ Phase I and/or Phase II metabolites
- % remaining and (if multiple timepoint) half-life of parent compound 
- Extracted ion chromatograms of parent and major metabolites 
- Proposed metabolic transformation for each observed metabolite 
- Tabulated list of metabolite masses, peak areas, and retention times 

Metabolite Identification Liver microsomes, S9, hepatocytes
✓ Phase I and/or Phase II metabolites

All of above plus: 

- MS/MS spectra of parent compound and observed metabolites
- Structural interpretation of productions of parent and metabolites 
- When possible, molecular formula and proposed structures of metabolites

In vivo Metabolite Analysis Plasma or tissue from in-life studies
✓ Circulating Phase I and Phase II metabolites
Either of above options


Waters Xevo G2 QTOF

  • Fast acquisition rates compatible with UHPLC separations
  • High resolution (> 20,000 FWHM) and accurate mass measurement (< 3 ppm)
  • MSE data acquisition to obtain data for precursor and product ions simultaneously

MetaboLynx software

  • Elemental composition of accurate mass measured analytes
  • Automated search for expected Phase I or Phase II metabolites
  • MassFragment tool for structural assignment of product ions  (fig.1.)
  • Mass defect filtering to identify metabolites based on accurate mass measurement of parent compound (fig. 2.)

Matrices for Metabolite Identification

  • Liver microsomes (with NADPH for oxidative Phase I metabolism or UDPGA for Phase II glucuronide conjugation)
  • Liver S9 (with NADPH for oxidative Phase I metabolism, or glutathione and UDPGA for Phase II glutathione conjugation and glucuronide conjugation, respectively)
  • Cryopreserved hepatocytes (Phase I and Phase II metabolism)
  • All matrices available for multiple species for cross-species metabolite profiling



Figure 1. Product ion data from high energy MS E data function can be queried with Mass Fragment tool to aid in structural assignment of fragment ions.


Figure 2. Mass defect filter can be applied to accurate mass data in order to isolate components (metabolites) which are chemically similar to parent compound.



1. FDA (2008) Guidance for Industry - Safety Testing of Drug Metabolites