The combination of LTQ-Orbitrap with reversed phase HPLC was successfully used by the group of Taguchi for the determination

of glycerophospholipids and PIP, either by full scan of molecular ions [53] or by a combination of high resolution precursor full scans and low resolution product ion scans [53,54]. Sato et al. even expanded this platform by separation of glycerophospholipids into three fractions with solid phase extraction (SPE) prior to LC-MS analysis [55]. Eicosanoids are an analytical challenge Inhibitors,research,lifescience,medical because they comprise a plethora of isomeric compounds. An excellent approach by reversed phase HPLC and LTQ-Orbitrap was shown by the group of Volmer [37], whereby retention time, exact mass and MS/MS fragment

intensities were all taken into account for successful identification of various isomers. A faster but somewhat less specific variant of this experimental setup is shown by the same group on an Orbitrap Exactive instrument coupled to reversed Inhibitors,research,lifescience,medical phase HPLC [56]. Although this instrumentation lacks any MS/MS capabilities, sub-ppm mass accuracies and retention time are sufficient for identification of certain glycerophospholipid classes in fast survey scans. 3.3. Digging Deeper into Structural Inhibitors,research,lifescience,medical Details Beyond the actual fatty acid composition of a lipid species, the fatty acid sn-position can be of importance for many biological functions. Most direct infusion and LC-MS methods in lipidomics are Inhibitors,research,lifescience,medical not able to separate lipid molecular species at the level of positional isomers. However, some very sophisticated chromatographic methods are able to obtain insight on lipid molecular geometry at this level. Separation of positional TG isomers was successfully achieved by using silver-ion HPLC [57]. This publication describes a

setup for serial connection of three HPLC columns to attain the necessary chromatographic resolution. Another excellent example of a Ku-0059436 mw highly specialized HPLC setup Resveratrol by the same Inhibitors,research,lifescience,medical group uses a 2D approach [58]. A first HILIC dimension separates and fractionates lipids by their respective lipid classes, and a second reversed phase dimension further separates lipid classes by their individual molecular composition. While these HPLC methods are very efficient and highly specific for separation of individual lipid molecular species, they are only suitable for a limited number of samples due to their long chromatographic separation times, particularly in 2D HPLC. Another highly specific LC-MS strategy is proposed by the group of Blair [59]. This experimental platform couples chiral normal phase HPLC to negative APCI-MS for separation and determination of regioisomeric and enantiomeric forms of eicosanoids.