This article is available online at http://www.jlr.orgLipids are the building blocks of all cell membranes and are thus essential for various biological functions varying from membrane traffi cking to signal transduction. Disorders in lipid metabolism play a key role in various diseases including cardiovascular disease, cancer, diabetes mellitus, and infl ammation ( 1, 2 ). Lipids have been classifi ed into eight categories (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides) by the International Committee for the Classifi cation and Nomenclature of Lipids in conjunction with the LIPID MAPS consortium, based on their chemically distinct functional backbones and hydrophobic nature ( 3, 4 ).As the study of lipids attracts more and more attention, lipidomics, as a branch of metabolomics, is a quickly emerging fi eld. It has been defi ned as "the full characterization of lipid molecular species and of their biological roles with respect to expression of proteins involved in lipid metabolism and function, including gene regulation" ( 5 ).In order to achieve the goal of full characterization, different MS-based approaches have been described. Some groups describe shotgun lipidomics using direct infusion into the mass spectrometer ( 6-11 ). Direct infusion MS has the advantage of short analysis times and consuming very small amounts of sample. However, structural elucidation of lipid isomers is not possible. In order to elucidate structures Abstract An ultraperformance LC (UPLC) method for the separation of different lipid molecular species and lipid isomers using a stationary phase incorporating charged surface hybrid (CSH) technology is described. The resulting enhanced separation possibilities of the method are demonstrated using standards and human plasma extracts. Lipids were extracted from human plasma samples with the Bligh and Dyer method. Separation of lipids was achieved on a 100 × 2.1 mm inner diameter CSH C 18 column using gradient elution with aqueous-acetonitrile-isopropanol mobile phases containing 10 mM ammonium formate/0.1% formic acid buffers at a fl ow rate of 0.4 ml/min. A UPLC run time of 20 min was routinely used, and a shorter method with a 10 min run time is also described. The method shows extremely stable retention times when human plasma extracts and a variety of biofl uids or tissues are analyzed [intra-assay relative standard deviation (RSD) <0.385% and <0.451% for 20 and 10 min gradients, respectively (n = 5); interassay RSD <0.673% and <0.763% for 20 and 10 min gradients, respectively (n = 30)]. The UPLC system was coupled to a hybrid quadrupole orthogonal acceleration time-of-fl ight mass spectrometer, equipped with a traveling wave ion-mobility cell. Besides demonstrating the separation for different lipids using the chromatographic method, we demonstrate the use of the ion-mobility MS platform for the structural elucidation of lipids. The method can now be used to elucidate structures of a wide variety of lipid...