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Quantitative Analysis of Lipids in Human Plasma Using Derivatization with H5/D5-Benzoyl Chloride and 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	Quantitative Analysis of Lipids in Human Plasma Using Derivatization with H5/D5-Benzoyl Chloride and RP-UHPLC/MS	Lipids play an important role in all organisms, such as membrane components, energy storage, precursors for metabolic processes, and signaling, which predestines lipids for biomarkers of cancer, cardiovascular diseases, and neurodegenerative diseases. The chemical derivatization of lipid functional groups can improve an extraction efficiency, chromatographic separation, and sensitivity. Benzoyl chloride is a non-hazardous derivatization agent with high reactivity for several functional groups producing esters with hydroxyl groups, amides with amino groups, and anhydrides with carboxylic acid groups. Mass spectrometry is a key technique in the lipidomic analysis and coupled to a separation method is typically used in lipidomics. Moreover, reversed-phase liquid chromatography enables to resolve isomeric lipids, such as the fatty acyl level and fatty acyl positions on the glycerol skeleton. Deproteinized human plasma was derivatized with benzoyl chloride in pyridine. The reaction mixture reacts 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 for non-targeted analysis and Xevo TQD for targeted analysis. Acquity UPLC BEH C18 column (150 mm×2.1 mm, 1.7 µm) was employed. The gradient elution using ACN/H2O (3:2, v/v) and IPA/ACN (9:1, v/v), both phases containing 8 mM ammonium formate and 0.1% formic acid, was set for the separation of lipid species. The reaction conditions, such as volume and ratio of reagents, reaction time, and temperature, were thoroughly optimized for 4 nonpolar and 8 polar lipid classes using spiked pooled plasma. The molar ratio 4:1 of pyridine and benzoyl chloride, and the reaction time of 60 min at ambient temperature provide the best yield. The reproducibility of reaction was investigated by 2 operators with reported RSD lower than 20 %. The derivatization and nonderivatization approaches were compare	Derivatization; Human plasma; Lipids