Metabolite identification
Metabolite identification (Met ID) is necessary throughout the drug discovery and development cycle. Often the question that needs to be answered in early drug discovery will be different from late drug discovery or development. 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 rigour.
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 characterisation 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.
A tiered approach to metabolite identification
Our tiered approach to metabolite identification describes study designs appropriate to the different points in a molecule’s development. These study designs are suggestions, and we are happy to discuss tailored experiments to answer specific questions to support the progress of your project.
The level of detail provided varies depending on the tier selected.
Tier 1 and 2 study designs are recommended for early discovery when trying to improve metabolic stability as part of the design, make, test cycle. This information aids the chemist with designing a more metabolically stable compound and is made available in a timely fashion.
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 experimental procedure and reporting for each tier are listed below.
You can also contact us at info@charnwood-molecular.com to discuss the option best suited to your project.
Met ID Tier 1 and Tier 2
For early discovery and to improve metabolic stability.
Experimental description
Identify major metabolites (maximum of three)
- Tier 1 from intrinsic clearance (CLint)sample in hepatocytes (time point dependent on CLint, compound concentration 1 µM).
- Tier 2, separate hepatocyte incubation (60/90-minute incubation, compound concentration 10 µM)
- MS/MS only on major metabolites
Report provided
Standard report format
- Summary table with proposed structures
- Details of LC method
Met ID Tier 3
Recommended for mid-stage discovery where a more detailed picture of the metabolic profile is needed but not necessarily full profile.
Experimental description
Identify major metabolites (>1% based on UV response or top 10 based on MS response) from separate incubation (90-minute incubation, compound concentration 10 µM)
- Targeted MS/MS carried out for structural ID
Report provided
Standard report format
- Summary table with proposed structures
- Glutathione or acyl glucuronides flagged
- Any human specific metabolites flagged (If possible)
- Additional minor metabolites are NOT listed
- Details of LC method
Met ID Tier 4
Recommended at late-stage discovery to compare metabolic profile of human with other species to determine metabolite coverage for possible safety studies
Experimental description
Cross species comparison of human profile with mouse/rat/dog/minipig/cynomolgus monkey hepatocytes
- Identify human metabolites (quantification based on UV response or MS response) from separate incubation (90-minute incubation, compound concentration 10 µM)
- Targeted MS/MS carried out for structural ID on all human metabolites
Report provided
Standard report format
- Summary table with proposed structures
- Any human specific or disproportionate metabolites flagged
- Glutathione or acyl glucuronides flagged
- Metabolites detected in other species but NOT in human are listed for information with no structural characterisation
- Details of LC method
Detailed report format (in addition to standard report)
- UV (where possible) and extracted ion chromatograms provided
- Full scan and product ion spectra included along with interpretation of key fragments
Met ID Tier 5
Recommended at late-stage discovery or development to identify metabolites from in vivo studies (plasma, urine or bile)
Experimental Description
Requirements will be discussed with clients prior to start but the normal format would be:
- Identify major circulating metabolites by using AUC pooled plasma/blood samples
- Proportionally pool urine/bile samples to provide one sample
- Identify major metabolites in urine/bile (≥1% based on UV or top 10 based on MS response)
- Minor metabolites are listed for information unless excessive numbers and discussed with client
- Targeted MS/MS carried out for structural ID on all significant metabolites
Report provided
Standard report format
- Summary table with proposed structures
- Glutathione or acyl glucuronides flagged
- Details of LC method
- UV (where possible) and extracted ion chromatograms provided
- Full scan and product ion spectra included along with interpretation of key fragments