Human in vitro liver metabolism using HLM, HLCYT and Liquid Chromatography coupled to High-Resolution Mass Spectrometry
Scope of the method
- Human health
- Basic Research
- In vitro - Ex vivo
- Human derived cells / tissues / organs
- Liquid chromatography
- mass spectrometry
- in vitro
- analytical chemistry
- liver metabolism
- Drug metabolism
- Drug discovery
A compound of interest (e.g. new psychoactive substance, endocrine disrupting compound, ...) is incubated with human liver microsomes and liver cytosolic fractions to generate both Phase I and II metabolites. Samples are prepared for analysis using a simple method in order to avoid possible losses of biotransformation products. The extracts are analysed using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Identification of the biotransformation products is performed using complementary screening workflows. These include a suspect screening based on in silico predictions and non-targeted screening using either vendor-specific or in-house developed open-source software protocols.
- - Warm water bath (37°C) ;
- - Temperature-controlled nitrogen evaporator ;
- - Centrifuge ;
- - LC coupled to high-resolution mass spectrometry (for identification).
- Published in peer reviewed journal
Pros, cons & Future potential
- - Optimized assay with different timepoints, negative and positive controls and method blanks ;
- - Tested for a variety of substrates (NPSs, EDCs, ...) resulting in multiple publications ;
- - Custom data analysis possible, according to research question ;
- - Besides analytical equipment (LC-HRMS) no need for expensive equipment.
- - Possible over or underestimation of in vivo biotransformation ;
- - Suspect screening dependent on strength of in silico predictions.
- No further optimizations are planned for the near future.
References, associated documents and other information
2019 - Vervliet - Toxicology - HLM DEMO.pdf
Contact personPhilippe Vervliet
OrganisationsUniversity of Antwerp
Department of Pharmaceutical Sciences