Optimisation of in vitro sample preparation for LC-MS metabolomics applications on HepaRG cell cultures

Abstract

The addition of stabilizers during liquid–liquid extraction improves the precision of untargeted LC-MS metabolomics workflows.

Optimization of a liquid chromatography-ion mobility-high resolution mass spectrometry platform for untargeted lipidomics and application to HepaRG cell extracts

“…The latter method replaces CHCl 3 for the less toxic MTBE, which also causes a change in the location of the solvent layers (the organic layer is the upper phase for Matyash's method) [10,75]. Two-phase extractions for a low amount of sample (e.g., 20 µL of plasma, 10 6 cells, 2.5 mg of tissue) have been successfully applied using the above-mentioned (adapted) Bligh and Dyer, and Matyash techniques to analyze each phase with different LC-MS methods selected based on the LogP of the metabolites [54,76,77]. The successful application of these methods shows that reproducible extractions for metabolomics can also be obtained with low sample amounts.…”

Exposure of HepaRG Cells to Sodium Saccharin Underpins the Importance of Including Non-Hepatotoxic Compounds When Investigating Toxicological Modes of Action Using Metabolomics

“…The cell cultures were harvested according to previously described protocols, full details are available in SM-3 [12,13]. Briefly, cells were prepared for extraction with a wash in phosphate buffered saline (37 °C) followed by freezing on liquid nitrogen.…”

Mass Spectrometry-Based Zebrafish Toxicometabolomics: A Review of Analytical and Data Quality Challenges

“…Complex organisms usually require more replicates than in vitro cell models to unveil meaningful biological information [ 44 , 54 ]. However, animals in controlled environments (e.g., standardized diet, day–night cycle times, temperature environment) require a smaller amount of replicates than epidemiological studies, but it is still advisable to keep the number of replicates to at least 6 per group [ 10 , 44 ].…”

“…The latter method replaces CHCl 3 for the less toxic MTBE, which also causes a change in the location of the solvent layers (the organic layer is the upper phase for Matyash’s method) [ 10 , 75 ]. Two-phase extractions for a low amount of sample (e.g., 20 μL of plasma, 10 6 cells, 2.5 mg of tissue) have been successfully applied using the above-mentioned (adapted) Bligh and Dyer, and Matyash techniques to analyze each phase with different LC-MS methods selected based on the LogP of the metabolites [ 54 , 76 , 77 ]. The successful application of these methods shows that reproducible extractions for metabolomics can also be obtained with low sample amounts.…”