Novel Application of Reversed-Phase UPLC-oaTOF-MS for Lipid Analysis in Complex Biological Mixtures: A New Tool for Lipidomics Paul D. Rainville,* ,† Chris L. Stumpf, † John P. Shockcor, † Robert S. Plumb, † and Jeremy K. Nicholson ‡ Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, and Biological Chemistry, Division of Biomedical Sciences, Sir Alexander Fleming Building, Imperial College London SW7 2AZ, United Kingdom Received November 18, 2006 Ultra-Performance LC (UPLC) utilizing sub-2-μm porous stationary phase particles operating with high linear velocities at pressures >9000 psi was coupled with orthogonal acceleration time-of-flight (oaTOF) mass spectrometry and successfully employed for the rapid separation of lipids from complex matrices. The UPLC system produced information-rich chromatograms with typical measured peak widths of 3 s at peak base, generating peak capacities in excess of 200 in 10 min. Further UPLC coupled with MS E technology provided parent and fragment mass information of lipids in one chromatographic run, thus, providing an attractive alternative to current LC methods for targeted lipid analysis as well as lipidomic studies. Keywords: Lipidomics • Metabolic Profiling • Ultra Performance LC • Mass Spectrometry Introduction Lipids are an important class of biomolecule that exist in great variety in higher organisms. The great diversity exhibited by these molecules is most probably due to the many bio- chemical functions in which these molecules are involved. Lipids are used as energy stores to fuel metabolism, as structural components in cell membranes, and are involved in many metabolic processes such as signal transduction, morphogenesis, secretion, and vesicle trafficking. 1,2 Metabolic diseases such as Diabetes mellitus have been shown to have a direct relationship with a disorder in the lipids and fatty acids that make up phospholipids, 3 a class of lipids that are defined by various specific polar head groups. This class of lipid has shown commercial use in biomembranes, skin care formula- tions, and in the making of liposomes, which are used in drug delivery as well as in cosmetics and detergents. 3 The analysis and profiling of lipids has therefore become increasingly important in the fields of food analysis, commercial applica- tions, and metabolic profiling. The ability to profile the lipids in biological fluids is also important, as it allows for the analysis of the effects of many candidate pharmaceuticals of metabolic pathways such as cholesterol synthesis. Lipidomics is a field of study that has rapidly expanded the study of lipids based on the advances made by electrospray ionization mass spec- trometry 4 that focuses on the study of lipid classes as they exist in their natural environment. The analysis of lipids has been performed by a diverse variety of approaches reflecting the diverse chemical subclasses. Gas chromatography (GC) and GC mass spectrometry (GC/MS) approaches provide a rapid, sensitive, and relatively cheap method of analysis, but require prior time-consuming chemical derivatization which can leads to inter laboratory variations. 5 1 H NMR provides a relatively fast method of profile analysis that can generate information on a range of lipids including lipoproteins, but the disadvantage of this approach is the difficulty in separating multiple overlapped lipidic species with the exception of lipoproteins. Lipids have been successfully analyzed by normal-phase high-performance liquid chroma- tography (HPLC) 6-8 and normal-phase HPLC coupled with mass spectrometry; 3,9-11 this method of analysis has high sensitivity and does not require any sample derivatization. However, the analysis times are typically long, 30-60 min, and peak resolution is typically poor, with peak width in the order of 20-30 s. Direct infusion of lipids into a mass spectrometer has also been shown as a method for characterization of cellular lipids, 4 but this type of analysis of simultaneously introducing multiple analytes into a mass spectrometer can affect the detection of low-abundance species due to ion suppression. Reversed-phase HPLC has been the technique of choice for both pharmaceutical and bioanalytical LC/MS/MS analysis. 12 This is due to the high efficiency of the separations, the compatibility of the mobile phase with biological and lipophilic samples, and the easy interfacing with a variety of detectors including UV, fluorescence, evaporative light scattering detec- tion, radio-chemical detection, mass spectrometry, and NMR. In fact, a majority of liquid chromatographic separations used today are based upon reversed-phase LC. However, as previ- ously stated, lipid analysis is often carried out using normal- phase chromatography and becomes difficult if one is switching between the two different modes of chromatography on a single system. The process of changing from reversed-phase to normal-phase chromatography entails replacing not only the * Corresponding author. E-mail: paul_rainville@waters.com. † Waters Corporation. ‡ Imperial College London. 552 Journal of Proteome Research 2007, 6, 552-558 10.1021/pr060611b CCC: $37.00  2007 American Chemical Society Published on Web 12/23/2006