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New derivatization method for the determination of non-polar lipids using RP-UHPLC/MS/MS in negative ion mode

Authors: Kadyrbekova Yasmin | Peterka Ondřej | Jirásko Robert | Holčapek Michal

Year: 2024

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	New derivatization method for the determination of non-polar lipids using RP-UHPLC/MS/MS in negative ion mode	Non-polar lipids are high abundant biomolecules found in cells and body fluids that play essential roles in a human organism. There are several classes that belong to non-polar lipids, such as sterols, prenols, and glycerolipids. One of the most abundant species of these lipids is cholesterol, from which other sterol metabolites can be formed, e.g., cholesterol esters, oxysterols, and bile acids. The concentrations of sterol and prenol metabolites in biological samples may be up to three orders of magnitude lower than cholesterol. Moreover, the low ionization efficiency, in-source fragmentation, and non-selective multiple reaction monitoring (MRM) transitions are typical for free sterols. Therefore, a derivatization method is needed to enhance their ionization properties using mass spectrometry (MS). The direct infusion MS is not preferred due to its lack of ability to separate isomeric and isobaric species, which can be resolved by liquid chromatography (LC). However, to achieve fast analysis and high resolution separation, the sub-2 µm particles columns are used. Reversed-phase (RP) chromatography is beneficial for the identification of lipid species and the combination with MRM transitions on QqQ instrument enables a highly sensitive quantitation of isomers. The new derivatization method using 3-(chlorosulfonyl)benzoic acid (Cl-SBA) has never been used before for the derivatization of sterols, prenols, and glycerolipids. The reaction enables the detection of these lipid classes in the negative ion mode, which minimizes the in-source fragmentation common in the positive ion mode, leading to higher sensitivity. The RP-UHPLC/MS/MS method was optimized based on 30 derivatized standards from 4 lipid classes (sterols, prenols, monoacylglycerols, and diacylglycerols). RP-UHPLC gradient (mobile phases A: ACN/H2O and B: ACN/IPA + both contained 0.1% formic acid and 5 mM ammonium formate) was improved resulting in shorter analysis time (20 min), where the Acquity UPLC BEH	Derivatization; Non-polar lipids; RP-UHPLC/MS/MS; negative ion mode