Why chromatography? That was the question Michal Holčapek sought to answer, specifically in relation to its use for the human lipidome.
In his keynote lecture at HPLC 2023, Holčapek presented an approach that involved unambiguous lipid identification, reliable quantitation, and tailored sample preparation protocols. His team’s goal was to create an atlas of the human lipidome, enabling future applications in the study and treatment of serious human diseases.
Holčapek focused on addressing the limitations of existing analytical workflows in accurately quantifying lipids and achieving sufficient coverage of the lipidome. To ensure unambiguous identification, high mass accuracy mass spectrometry (MS) and MS/MS techniques, utilizing both positive and negative ion modes, were employed. The characteristic fragmentation behaviour and retention dependences for carbon number and double bond number were also considered, enabling accurate lipid identification.
For reliable quantitation, the analytical methods were validated, and appropriate exogenous internal standards and quality control samples were employed. This approach ensured lipid concentration measurements were accurate, thus providing a solid foundation for lipidomics research.
Holčapek also emphasized the importance of data reporting in lipidomics. He proposed lipid level consistent with experimental data, shorthand nomenclature, and standardized reporting formats to facilitate data interpretation and comparison across studies.
To achieve comprehensive coverage of the lipidome, various chromatographic modes were employed. Ultrahigh-pressure liquid chromatography (UHPLC) coupled with MS, as well as ultrahigh-pressure supercritical fluid chromatography (UHPSFC) coupled with MS, were used. These chromatographic approaches allowed for the separation of different lipid classes and lipid species, including hydrophilic interaction chromatography (HILIC)-like mode and reversed-phase-like mode separations.
The proposed methods aim to enable the reliable quantitation of over 1000 lipid species in human lipidomics, with a particular focus on human plasma. By building an atlas of the human lipidome, researchers can gain a deeper understanding of lipid-related processes in health and disease. This knowledge holds the potential for future applications in the diagnosis, treatment, and prevention of serious human diseases, particularly those with lipid metabolism implications, such as cancer.
(1) Holčapek, M. Potential of Various Chromatographic Modes for Comprehensive Coverage of Human Lipidome. Presented at: HPLC 2023. June 18–22, 2023. Duesseldorf, Germany. KN22.