Tailored liquid chromatography-mass spectrometry analysis improves the coverage of the intracellular metabolome of HepaRG cells.
Metabolomics protocols are often combined with Liquid Chromatography-Mass Spectrometry (LC-MS) using mostly reversed phase chromatography coupled to accurate mass spectrometry, e.g. quadrupole time-of-flight (QTOF) mass spectrometers to measure as many metabolites as possible. In this study, we optimised the LC-MS separation of cell extracts after fractionation in polar and non-polar fractions. Both phases were analysed separately in a tailored approach in four different runs (two for the non-polar and two for the polar-fraction), each of them specifically adapted to improve the separation of the metabolites present in the extract. This approach improves the coverage of a broad range of the metabolome of the HepaRG cells and the separation of intra-class metabolites. The non-polar fraction was analysed using a C18-column with end-capping, mobile phase compositions were specifically adapted for each ionisation mode using different co-solvents and buffers. The polar extracts were analysed with a mixed mode Hydrophilic Interaction Liquid Chromatography (HILIC) system. Acidic metabolites from glycolysis and the Krebs cycle, together with phosphorylated compounds, were best detected with a method using ion pairing (IP) with tributylamine and separation on a phenyl-hexyl column. Accurate mass detection was performed with the QTOF in MS-mode only using an extended dynamic range to improve the quality of the dataset. Parameters with the greatest impact on the detection were the balance between mass accuracy and linear range, the fragmentor voltage, the capillary voltage, the nozzle voltage, and the nebuliser pressure. By using a tailored approach for the intracellular HepaRG metabolome, consisting of three different LC techniques, over 2200 metabolites can be measured with a high precision and acceptable linear range. The developed method is suited for qualitative untargeted LC-MS metabolomics studies.
Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk-Antwerp, Belgium. Electronic address: Matthias.Cuykx@uantwerpen.be.
Matthias
M
Cuykx
Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk-Antwerp, Belgium.
Noelia
N
Negreira
Department of Mathematics & Computer Science, University of Antwerp, Middelheimlaan 1, 2020 Antwerp, Belgium.
Charlie
C
Beirnaert
Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk-Antwerp, Belgium.
Nele
N
Van den Eede
Department of In Vitro Toxicology and Dermato-Cosmetology, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Belgium.
Robim
R
Rodrigues
Department of In Vitro Toxicology and Dermato-Cosmetology, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Belgium.
Tamara
T
Vanhaecke
Department of Mathematics & Computer Science, University of Antwerp, Middelheimlaan 1, 2020 Antwerp, Belgium.
Kris
K
Laukens
Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk-Antwerp, Belgium. Electronic address: Adrian.Covaci@uantwerpen.be.
Adrian
A
Covaci
D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*
HepaRG; Hydrophilic liquid interaction chromatography; Ion pairing; Liquid chromatography–Mass spectrometry; Metabolomics; Optimization