In Vitro Metabolite Profiling and Structure Identification

Background and Service Details:

Enzymatic biotransformation of drugs in living systems strongly affect their biological activity, which sometimes results in metabolite with decreased bioavailability and enhanced toxicity. The knowledge of specific sites of metabolic transformations is useful for guiding synthetic optimization of the lead compounds or drug candidates to overcome the stability and toxicity issues.

To elucidate the main routes of hepatic metabolism, incubation of test compounds is carried out with liver microsomes (mouse, rat or human), that provide a wide range of the metabolizing enzymes. Additionally, S9 fractions from mouse, rat or and human liver can be used for a broader coverage of possible biotransformations (both phases I and II of metabolism). Some compounds are unstable in blood plasma, therefore plasma stability test can be included in the service. A typical protocol includes incubation of the test compound  with microsomes  or S9 in the presence of co-factors, sampling at 2 time-points (0 and 60 minutes), reaction quenching and centrifugation. The analysis of the incubation sample and comparison to the control (quenched at 0 time) is performed using liquid chromatography/tandem mass spectrometry.

Our approach to drug metabolite identification integrates multiple reaction monitoring of predicted metabolites with information dependent acquisition (MRM-IDA) using hybrid triple quadrupole linear ion trap mass spectrometer API5000 QTRAP (AB Sciex). The multiple reaction monitoring with information dependent acquisition MRM-IDA methods are created using LightSight 2.3 software (AB Sciex) comprising a comprehensive database of all classical metabolic biotransformations, both phases I and II of metabolism. The methods comprise the survey MRM-IDA scans for parent compound and metabolites linked to information dependent enhanced product ion (EPI) scan. The fragmentation pathways of parent compound and metabolites are elucidated by analysis of MS/MS product ion spectra obtained from enhanced product ion (EPI) scans. The identification of metabolites and assignment of MS/MS product ions with specific fragments of molecule is performed using ACD/Labs MS Fragmenter software.


Data include ion chromatograms of parent compound and metabolites, the table containing metabolites references and where possible molecular formulae, types of biotransformations, masses, m/z, mass differences from the parent, and retention times. The structure identification data comprise MS/MS spectra and details of the product ion fragments for parent compound and metabolites, as well as structural assignment based on the key fragments observed. Finally, we provide a detailed report containing proposed metabolites structures and expected metabolic pathways.

Sample Submission:

A minimal accurately weighable quantity of dry compound (~1 mg or 2 µmol) or 50 µL of 10-20 mM stock DMSO solution is required for this assay. For multiple assays, smaller amount of compound per assay may be sufficient, which should be discussed for each particular project. Molecular structures are necessary for metabolite profiling studies.