Comparison of reversed-phase, hydrophilic interaction, and porous graphitic carbon chromatography columns for an untargeted toxicometabolomics study in pooled human liver microsomes, rat urine, and rat plasma

• Published in: Metabolomics Vol. 20; no. 3; p. 49
• Main Authors: Hemmer, Selina, Manier, Sascha K., Wagmann, Lea, Meyer, Markus R.
• Format: Journal Article
• Language: English
• Published: New York Springer US 30-04-2024; Springer Nature B.V
• Subjects: Ammonium; Animals; Biochemistry; Biomedical and Life Sciences; Biomedicine; Carbon; Cell Biology; Chromatography; Chromatography, Liquid - methods; Chromatography, Reverse-Phase - methods; Developmental Biology; Graphite - chemistry; Humans; Hydrophobic and Hydrophilic Interactions; Investigations; Life Sciences; Liquid chromatography; Liver; Mass spectroscopy; Metabolome; Metabolomics; Metabolomics - methods; Microsomes; Microsomes, Liver - metabolism; Molecular Medicine; Original; Original Article; Plasma - chemistry; Plasma - metabolism; Porosity; Rats; Sulfonic acid; Urine; Quality assurance; Hydrophilic interaction chromatography columns; LC-HRMS; Reversed-phase columns; Untargeted metabolomics
• ISSN: 1573-3890; 1573-3882; 1573-3890
• DOI: 10.1007/s11306-024-02115-0

Introduction
Untargeted metabolomics studies are expected to cover a wide range of compound classes with high chemical diversity and complexity. Thus, optimizing (pre-)analytical parameters such as the analytical liquid chromatography (LC) column is crucial and the selection of the column depends primarily on the study purpose.
Objectives
The current investigation aimed to compare six different analytical columns. First, by comparing the chromatographic resolution of selected compounds. Second, on the outcome of an untargeted toxicometabolomics study using pooled human liver microsomes (pHLM), rat plasma, and rat urine as matrices.
Methods
Separation and analysis were performed using three different reversed-phase (Phenyl-Hexyl, BEH C
18
, and Gold C
18
), two hydrophilic interaction chromatography (HILIC) (ammonium-sulfonic acid and sulfobetaine), and one porous graphitic carbon (PGC) columns coupled to high-resolution mass spectrometry (HRMS). Their impact was evaluated based on the column performance and the size of feature count, amongst others.
Results
All three reversed-phase columns showed a similar performance, whereas the PGC column was superior to both HILIC columns at least for polar compounds. Comparing the size of feature count across all datasets, most features were detected using the Phenyl-Hexyl or sulfobetaine column. Considering the matrices, most significant features were detected in urine and pHLM after using the sulfobetaine and in plasma after using the ammonium-sulfonic acid column.
Conclusion
The results underline that the outcome of this untargeted toxicometabolomic study LC-HRMS metabolomic study was highly influenced by the analytical column, with the Phenyl-Hexyl or sulfobetaine column being the most suitable. However, column selection may also depend on the investigated compounds as well as on the investigated matrix.
Graphical abstract