Human in vitro liver metabolism using HLM, HLCYT and Liquid Chromatography coupled to High-Resolution Mass Spectrometry

Scope of the method

Alternative method relates to
  • Environment
  • Human health
Alternative method is situated in
  • Basic Research
Type of alternative method
  • In vitro - Ex vivo
This method makes use of
  • Human derived cells / tissues / organs
Specify the type of cells/tissues/organs
Human Liver Microsomes and Human Liver Cytosol

Description

Method keywords
  • HLM
  • HLCYT
  • Liquid chromatography
  • mass spectrometry
  • Metabolism
  • liver
  • in vitro
Scientific area keywords
  • Toxicology
  • analytical chemistry
  • liver metabolism
  • Drug metabolism
  • Drug discovery
Method description

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.

Lab equipment
  • - Warm water bath (37°C)
  • - Temperature-controlled nitrogen evaporator
  • - Centrifuge
  • - LC coupled to high-resolution mass spectrometry (for identification)
Method status
  • Published in peer reviewed journal

Pros, cons & Future potential

Advantages
  • - 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
Challenges
  • - Possible over or underestimation of in vivo biotransformation
  • - Suspect screening dependant on strength of in silico predictions
Modifications

- No further optimizations are planned for the near future.

References, associated documents and other information

Associated documents
2018 - Vervliet Mortele et al - DTA - 5Cl-THJ-018.pdf 2019 - Vervliet - Toxicology - HLM DEMO.pdf

Contact person

Philippe Vervliet

Organisations

University of Antwerp
Department of Pharmaceutical Sciences
Belgium