Abstract
In respect of the manifold involvement of lipids in biochemical processes, the analysis of intact and underivatised lipids of body fluids as well as cell and tissue extracts is still a challenging task, if detailed molecular information is required. Therefore, the advantage of combined use of high-pressure liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy will be shown analyzing three different types of extracts of the ubiquitous membrane component phosphatidylcholine. At first, different reversed phase modifications were tested on phosphatidylcholines (PC) with the same effective carbon number (ECN) for their applicability in lipid analysis. The results were taken to improve the separation of three natural PC extract types and a new reversed phase (RP)-HPLC method was developed. The individual species were characterized by one- and two-dimensional NMR and positive or negative ion mode quadrupole time of flight (q-TOF)-MS as well as MS/MS techniques. Furthermore, ion suppression effects during electrospray ionisation (ESI), difficulties, limits, and advantages of the individual analytical techniques are addressed.
Highlights
The analysis of native and underivatized lipids within body fluids as well as cell and tissue extracts is still a challenging task, in particular, if the molecular structure of individual components needs to be identified in decently short time
high-pressure liquid chromatography (HPLC) offers the separation of lipid classes using the normal phase mode (NP) and the separation according to the different fatty acid residues of an individual lipid in the reversed phase mode (RP)
This paper presents an efficient RP-HPLC setup to separate phosphatidylcholines, which will be extendable to separate other polar phospholipids
Summary
The analysis of native and underivatized lipids within body fluids as well as cell and tissue extracts is still a challenging task, in particular, if the molecular structure of individual components needs to be identified in decently short time. Numerous studies dealt with PCs in the past because of their utmost biochemical and clinical importance and many different analytical techniques have been proposed to get an insight into metabolic turnover or to characterize pathophysiological deviations of the native lipid composition Most of these techniques suffer from various drawbacks as being time-consuming, insensitive, destructive, or not related to individual substructures. HPLC offers the separation of lipid classes using the normal phase mode (NP) and the separation according to the different fatty acid residues of an individual lipid in the reversed phase mode (RP). In this case, a successful separation depends distinctly on the appropriate selection of the stationary phase. Only minor information is obtained with respect to the fatty acid residues. 1HNMR measurements are widely used, as they contain more information about the fatty acids in general, but the connection to the glycerol backbone is missing due to massive signal overlap. 2D-NMR involving the 13C nucleus provides a lot more resolution and more information about individual species, but the low NMR sensitivity of the 13C isotope prevents a fast and wide application of this technique in a routine analysis [44–48]
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