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Metabolite Identification and Profiling

In vitro metabolism in hepatocytes, liver microsomes, S9, recombinant enzymes or extrahepatic tissues 
• Small drug-like compounds and peptides
• UPLC with Q-TOF-MS or QE-Orbitrap-MS and UV detection
• UPLC with on-line radiodetection
• Isolation of metabolites and structural elucidation by NMR

Metabolite identification and profiling are relevant in several stages of drug discovery & development. Optimisation of metabolic clearance requires information on the main metabolically labile “soft-spots” and their further chemical modification. Selection of suitable animal species for toxicity studies is facilitated by the prediction of species with most similar metabolite profiles with respect to human (first in vitro and then in vivo). The correct evaluation of metabolic enzymes involved in the clearance requires also identification of the metabolites, and eventually the metabolic clearance routes in human in vivo has to be characterised in a detailed level.

For in vitro this typically requires studies with enzyme sources such as liver (or extrahepatic) microsomes, S9 fraction, hepatocytes, and recombinant enzymes, while for in vivo the plasma and excreta are the major sample types. Analytically the UPLC/high-resolution-MS/MS techniques are the major tool for metabolite identification in all study types, supported partially by additional detection technologies such as UV/PDA, or radiodetection for the studies where 3H or 14C-labeled compounds are used. Modern NMR spectroscopy after upscaled production and isolation of the metabolite is also a valuable tool for both qualitative and quantitative purposes. For peptides, the metabolism (catabolism) is most often based on amide/peptide-bond hydrolysis reactions, which are efficiently identified by UPLC/high-resolution MS techniques.