Strukturelle und quantitative Identifizierung der Einzelkomponenten in Lipidgemischen

Abstract

Lipidomics aims to qualitatively and quantitatively define lipid classes, including their molecular species, in biological systems. Continuous technical advances in instrumentation enable a high level of sensitivity and precision. Due to the broad diversity of chemical structures of lipids, different methods and strategies are being applied for the investigation of lipids. In this study lipophilic extracts of pig brain (liver, kidney, heart and human blood plasma) were examined with Nuclear Magnetic Resonance (NMR) spectroscopy and Mass Spectrometry (MS) after separation with solid phase extraction (SPE) in different lipid fractions. The aim of this work was the identification and quantification of the individual components in lipid mixtures. After the class separation the individual components of phosphatidylethanolamine and galactosylcerebroside were further separated with an isocratic High Performance Liquid Chromatography(HPLC)method. At first an optimized dual phase extraction procedure for tissues was developed in this thesis which enables a quantitative isolation of analytes under mild conditions. The focus of this work was the SPE method development. The SPE technique is easy, rapid, and reliable. The growing popularity of SPE is in part due to the operational simplicity and cost reduction in solvents and because it is easy to automate. The developed SPE method for phospholipid class separation with the use of silica gel cartridge allowed the separation of neutral lipids, galactocerebroside, sulfatide, cardiolipin, phosphatidylethanolamine and plasmalogen, as well as the approximately separation of phosphatidylcholine and sphingomyeline. The method proved to be reproducible and showed recoveries higher 73%. The individual components of the phosphatidylethanolamine fraction from each tissue were analyzed with the separation by RP-HPLC-MS. A total of 25 phosphatidylethanolamine components were baseline separated in a period of 20 min (eluting 6.8 min - 18.6 min). Here also the differences of the lipid components of the various organs were analyzed and discussed in relation to their functions. With the same method 19 galactocerebroside were separated. This confirms the potential of the method developed in this work for the detailed characterization of heterogeneous lipid compositions.