Automated Sample Preparation and Data Collection Workflow for High-Throughput In Vitro Metabolomics

Area of Science:

• Toxicology and Pharmacology
• Analytical Chemistry
• Biotechnology

Background:

• Regulatory bodies increasingly value in vitro screening and metabolomics as new approach methodologies (NAMs) for chemical risk assessments.
• A key challenge in high-throughput in vitro toxicometabolomics is automating sample preparation for low biomass samples.

Purpose of the Study:

• To develop, characterize, and demonstrate an automated sample preparation and analysis workflow for in vitro metabolomics.
• To assess the repeatability and throughput of the automated workflow for HepaRG cells using mass spectrometry.

Main Methods:

• Utilized a Biomek i7 Hybrid Workstation for automated sample preparation in 96-well microplates.
• Employed Orbitrap Elite high-resolution nanoelectrospray direct infusion mass spectrometry (nESI-DIMS) for polar metabolite and lipid analysis.
• Evaluated experimental conditions including extraction timing, sample order, microplate position, and well location.

Main Results:

• Demonstrated good repeatability of the workflow with a median relative standard deviation (mRSD) below 30%.
• Achieved high repeatability across a large cohort study (4860 samples) after method optimization.
• Significantly improved sample throughput for in vitro metabolomics analysis.

Conclusions:

• The automated in vitro metabolomics workflow is repeatable and suitable for high-throughput screening.
• This workflow advances the application of metabolomics within NAMs for chemical safety and risk prioritization.
• The developed method addresses the challenge of automated sample preparation for low biomass in vitro screening.