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H/D Pooling of Benzoyl Derivatives: Improvement of quantitative analysis and lipidomic profiling using RP-UHPLC/MS

Authors: Peterka Ondřej | Jirásko Robert | Vaňková Zuzana | Idkowiak Jakub | Holčapek Michal

Year: 2022

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	H/D Pooling of Benzoyl Derivatives: Improvement of quantitative analysis and lipidomic profiling using RP-UHPLC/MS	Lipids play an important role in all organisms, such as membrane components, energy storage, and signaling, which predestines lipids for biomarkers of cancer, cardiovascular diseases, and neurodegenerative diseases. Mass spectrometry combined with separation techniques is a key approach for the lipid analysis, which result in the identification and quantitation of large number of lipids. Reversed-phase liquid chromatography enables resolving isomeric lipids, but a large number of internal standards must be used for accurate quantitation. In this work, we present our derivatization approach providing an internal standard per each derivatized lipid due to the coelution of analyte and internal standard. Deproteinized human plasma was derivatized with benzoyl chloride in pyridine. The reaction mixture reacted at ambient temperature for 60 minutes. The reaction was stopped by applying a modified Folch lipid extraction protocol. We used UHPLC connected to high-resolution mass spectrometer Xevo G2-XS QTOF (Waters) for non-targeted analysis and QTRAP 6500 (Sciex) for targeted lipidomic analysis. Acquity UPLC BEH C18 column (150 mm × 2.1 mm, 1.7 μm) was employed. The derivatization method was optimized using the human plasma spiked with 22 internal standards. The new method reports high reproducibility, repeatability (RSD <15%), significantly higher sensitivity, and lower LOD in comparison with non-derivatization approach. H/D pooling means the mixing of non-labelled (H) and labelled (D) derivatives in the same ratio, which leads to the creation of doublets of analyte and internal standard, which simplifies the identification and provides internal standard per each derivatized compound with the same matrix effect and ionization efficiency. We identified 297 lipid species from 11 lipid classes based on the highly confident identification of lipids in human serum (MRM transition, retention dependences, and H/D doublets). This approach was fully validated and 159 lipid species	Lipidomics; Derivatization; Human serum; Pancreatic cancer