Nevertheless, in cases of a defined number of lipids, this can be a highly specific identification strategy as shown successfully for oxysterols

[42], positional isomer analysis of phospholipids [43] and in depth analysis of PIP species [44]. While chromatographic separation of lipids is often performed on reversed phase HPLC according to fatty acyl Inhibitors,research,lifescience,medical chains, this strategy runs into its limits when cholesterol esters (CE) are to be analyzed in the presence of bulk amounts of TG. Due to their very similar hydrophobicity CE and TG are hardly separated on reversed phase and hydrophilic interaction liquid chromatography (HILIC) columns, resulting in suppression of low abundant CE by TG (Figure 2). In contrast, silica-based normal phase HPLC provides separation of these lipid classes by their polar functional groups, but usually highly non polar solvents with low ionization capacity have to be used. Hutchins et al. [45] use APCI and post-column addition of a polar solvent to increase ionization properties of the non polar solvent Inhibitors,research,lifescience,medical eluting Inhibitors,research,lifescience,medical from normal phase HPLC. This results in a practicable online bridging between normal phase HPLC and triple quadrupole mass spectrometry, which can either be used in precursor ion, MRM or single quadrupole mode for determination of neutral lipids [34,46]. 3.2. High

Resolution Mass Spectrometry Quadrupole-TOF mass spectrometry offers several Inhibitors,research,lifescience,medical advantages. On one hand, this instrumentation provides resolution of up to 40,000

and mass accuracy of better than 5 ppm, which is sufficient for pinning down many of the elemental compositions encountered in lipidomic analysis. On the other hand, TOF analyzers have a very high scan rate and acquire full product ion spectra very fast and efficient. On the Inhibitors,research,lifescience,medical downside is the usually limited dynamic range of the detector, which limits quantitation to a rather narrow concentration range. Nevertheless this kind of instrumentation is a valuable tool when coupled to reversed phase HPLC. Successful application of this experimental setup was used for analysis of TG and oxidized TG species. In this case, it was even possible to determine the actual fatty acid composition of TG molecular Casein kinase 1 species by product ion spectra on all major species [47]. An excellent example for an integrated lipidomic platform relying on reversed phase ultra performance liquid chromatography (UPLC) quadrupole-TOF is shown by the group of Oresic [48], whereby a Rapamycin combination of retention time, exact precursor mass and product ion spectra are used for identification of lipids from various lipid classes. In contrast to widely used gradient elution, the group of Wenk present a profiling method based on quadrupole-TOF and isocratic reversed phase HPLC [49] used for determination of anionic glycerophospholipids, glycolipids, fatty acids, prenols and sphingolipids.