Metabolite Identification (Met ID)

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Metabolite identification (Met ID) is key to understanding the metabolic fate of a potential drug candidate and is an important part of the drug discovery and development process. However, the reasons for doing this change throughout the life of a drug discovery campaign.

Depending on the stage a molecule is at, it is appropriate to tailor the approach to metabolite identification both in terms of sample analysis and degree of data processing rigor.

In early discovery, the objective is to design potent, metabolically stable compounds and knowing the major route of metabolism quickly can help the chemists achieve this as part of the design-make-test-analyse cycle.

At later stages, however, it may be more relevant to compare full metabolic profiles in human with other species to determine metabolite coverage for possible safety studies.

Combining Technology & Expertise

Our expert team use the Thermo ScientificTM Orbitrap Exploris 120 mass spectrometer with their Vanquish Horizon ultra-high performance liquid chromatography (UHPLC) system.

This enables us to offer metabolite identification using the high-resolution capability of the instrument to provide accurate mass data to aid the structural characterization of metabolites.

Using their combined experience of over 60 years, our team is able to provide you with analysis and insight to progress your project.

Metabolic Profiling Services

A Tiered Approach to Metabolite Identification (MetID)

Our tiered approach to metabolite identification allows studies to be designed that are appropriate to the different stages in a molecule’s development. The study designs provided below are suggestions, and we are happy to discuss tailored experiments to answer specific questions.

The level of detail provided varies depending on the tier of study design selected.

Tiers 1 and 2 are recommended for early discovery when trying to improve metabolic stability. This information aids the chemist with designing a more metabolically stable compound as part of the design-make-test-analyse cycle.

Tiers 3, 4 and 5 are aimed at the later stages in the discovery and development process and provide a more detailed report, e.g. in Tier 4, a comparison of the metabolic profile of human with other species is provided.

Full details of methodology and reporting for each tier are listed below. 

You can also contact us to discuss the option best suited to your project.

For early discovery and to highlight metabolic liabilities.

Methodology

Identify major metabolites (maximum of three)

  • Tier 1 from intrinsic clearance (CLint) sample in microsomes or hepatocytes
    • Time point dependent on CLint, compound concentration 1 µM
  • Tier 2, separate microsome or hepatocyte incubation
    • 60 or 90-minute incubation, compound concentration 10 µM
  • MS/MS only on major metabolite

Reporting

Standard report format:

  • Summary table with proposed structures
  • Details of LC-MS method

Understanding the metabolic fate of a potential drug candidate is an important part of the drug discovery and development process but the reasons for doing this change throughout the life of a drug discovery campaign.

Recommended for mid-stage discovery where a more detailed picture of the metabolic profile is needed but not necessarily full profile.

Methodology

Identify major metabolites (>1% based on UV response or top 10 based on MS response) from a separate incubation (90-minute hepatocyte incubation, compound concentration 10 µM)

  • Targeted MS/MS carried out for structural ID

Reporting

Standard report format:

  • Summary table with proposed structures
  • Any human specific metabolites flagged (if possible)
  • Additional minor metabolites are NOT listed
  • Details of LC-MS method

Recommended at late-stage discovery to compare the metabolic profile in human with other species to determine metabolite coverage for possible safety studies.

Methodology

Cross species comparison of human profile with other pre-clinical species hepatocytes

  • Identify human metabolites (quantification based on UV or MS response) from a separate incubation (90-minute hepatocyte incubation, compound concentration 10 µM)
  • Targeted MS/MS carried out for structural ID on all human metabolites

Reporting

Standard report format:

  • Summary table with proposed structures
  • Any human specific or disproportionate metabolites flagged
  • Metabolites detected in other species but NOT in human are listed for information with no structural characterization
  • Details of LC-MS method

Detailed report format (available upon request):

  • UV (where possible) and extracted ion chromatograms provided
  • Full scan and product ion spectra included along with interpretation of key fragments

Recommended at late-stage discovery or development to identify metabolites from in vivo studies (e.g., plasma, urine or bile).

Methodology

Requirements will be discussed with clients prior to study start, but the typical format would be as follows:

  • Identify major circulating metabolites from AUC pooled plasma/blood samples
  • Proportionally pool other samples and identify major metabolites in (≥1% based on UV or top 10 based on MS response)
  • Minor metabolites are listed as appropriate
  • Targeted MS/MS carried out for structural ID on all significant metabolites

Reporting

Standard report format:

  • Summary table with proposed structures
  • UV (where possible) and extracted ion chromatograms provided
  • Full scan and product ion spectra included along with interpretation of key fragments
  • Details of LC-MS method

ADME / DMPK