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

Untargeted metabolomics studies are designed to observe as many metabolites as possible. A reliable protocol should be fast, efficient, unspecific and it should introduce as little variance as possible. Liquidliquid extraction (LLE) is often applied during sample preparation, since it leads to a polar (aqueous) and a non-polar (solvent) fraction. In the present study, we have applied for the first time pH buffers and other additives during the LLE to prevent degradation of the metabolites and to improve the precision of the method. The use of chamber slides in comparison to well plates improved the washing and quenching step and reduced the average quench time. Extraction efficiency and usability improved as well. The stabilisation of the pH of the LLE-solvent and the addition of anti-oxidants and chelators improved the precision of the method. Optimal results were observed at buffer strength of 10 mM at pH 4.4. Ascorbic acid (0.5 mM) and butyl-hydroxytoluene (1 mM) in combination with EDTA (1 mM) prevented oxidation and degradation.