Quantitative lipidomics: High-throughput analysis of clinical samples of body fluids

Abstract

Lipidomics is a subgroup of metabolomics, which is the last step of the omics cascade closest to the organism function, and therefore the analysis of metabolites (including lipids) can provide an important information about ongoing processes in the human body, such as diseases or disorders reflected in dysregulations of lipid concentrations. The main goal of this Ph.D. thesis is the development of high-throughput methods for the quantitation of lipids in biological samples using the following mass spectrometry (MS) based strategies: lipid species separation and lipid class separation, which have own requirements for the lipid quantitation mainly based on the selection and the number of internal standards (IS). The lipid species separation is applied for the analysis of oxylipins using a reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) coupled to tandem mass spectrometry with prior solid phase extraction from biological samples. The lipid class separation is used for the comprehensive quantitation of multiple lipid classes including monoacylglycerols, diacylglycerols, triacylglycerols, cholesteryl-esters, ceramides, sphingomyelins, phosphatidylcholines, and lysophosphatidylcholines. The lipid class separation approach is achieved by hydrophilic interaction ultrahigh-performance liquid chromatography (HILIC-UHPLC) or ultrahighperformance supercritical fluid chromatography (UHPSFC) coupled to mass spectrometry. Advantages and limitations of both approaches for the lipidomic quantitation are discussed including the method validation, the use of quality control samples, and the data normalization using NIST SRM 1950 Reference Material human plasma. Finally, all developed analytical methods have been applied for sets of clinical samples of cancer patients and healthy controls followed by multivariate data analysis to distinguish both groups.