Author's personal copy Sphingolipid profiling of human plasma and FPLC-separated lipoprotein fractions by hydrophilic interaction chromatography tandem mass spectrometry Max Scherer, Alfred Böttcher, Gerd Schmitz, Gerhard Liebisch ⁎ Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany abstract article info Article history: Received 27 August 2010 Received in revised form 25 October 2010 Accepted 8 November 2010 Available online 23 November 2010 Keywords: Lactosylceramide Sphingoid base Glycosylceramide High throughput Lipidomics Sphingolipidomics Sphingolipids comprise bioactive molecules which are known to play important roles both as intracellular and extracellular signalling molecules. Here we used a previously developed hydrophilic interaction chromatog- raphy tandem mass spectrometry (HILIC-MS/MS) method to profile plasma sphingolipids. Method validation showed sufficient precision and sensitivity for application in large clinical studies. Sample stability testing demonstrated that immediate plasma separation is important to achieve reliable results. Analysis of plasma from 25 healthy blood donors revealed a comprehensive overview of free sphingoid base, sphingosylpho- sphorylcholine (SPC), hexosylceramide (HexCer), lactosylceramide (LacCer), and ceramide-1-phosphate (Cer1P) species level. Besides the major sphingoid base sphingosine (d18:1), we found d16:1 and d18:2 species in most of these lipid classes. Interestingly, pronounced differences were detected in the species profiles of HexCer and LacCer. Additionally, sphingolipids were quantified in lipoprotein fractions prepared by fast performance liquid chromatography (FPLC). HexCer and LacCer showed similar distributions with about 50% in LDL, 40% in HDL and less than 10% in the VLDL fraction. More than 90% of sphingoid base phosphates were found in HDL and albumin containing fractions. In summary, HILIC-MS/MS provides a valuable tool to profile minor sphingolipid species in plasma and in lipoprotein fractions. Comparing profiles from tissues or blood cells, these species profiles may help to address the origin of plasma sphingolipids. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Sphingolipids comprise bioactive molecules [1–7] that influence various cellular functions including growth arrest, apoptosis, prolifer- ation and differentiation. Besides their intracellular signalling functions, sphingolipids act extracellularly by binding to specific receptors. They play important roles in several diseases including cancer, vascular and metabolic diseases [1,5,6,8–10]. A number of publications investigated plasma level of sphingomy- elin, ceramide species and sphingosine-1-phosphate as well as their biological function and disease association [3,11–19]. Apart from two recent publications [20,21], only limited information is available on the occurrence of minor sphingolipid species in human plasma. Recently, we developed a fast and reliable liquid chromatography- tandem mass spectrometry (LC-MS/MS) method for sphingolipid profiling based on hydrophilic interaction chromatography (HILIC) which allows the simultaneous quantification of sphingoid bases and their methylated products, sphingosylphosphorylcholine (SPC), hexosylceramide (HexCer), lactosylceramide (LacCer) and ceramide- 1-phosphate (Cer1P) species [22]. Aim of this study was to provide a detailed sphingolipid pattern of human plasma and lipoprotein fractions. Therefore, we analyzed sphingolipid species of human plasma from healthy blood donors by HILIC-MS/MS [22,23]. Additionally, we analyzed the sphingolipid class and species composition of lipoprotein fractions separated by fast performance liquid chromatography (FPLC) [19]. 2. Materials and methods 2.1. Subjects Blood samples were obtained from healthy normo-lipidemic vol- unteers. Informed consent and approval of the Hospital Ethics Com- mittee were obtained. Biochimica et Biophysica Acta 1811 (2011) 68–75 Abbreviations: CE, collision energy; Cer1P, ceramide-1-phosphate; CV, coefficient of variation; DimetSPH, dimethyl-sphingosine; ESI-MS/MS, electrospray ionization tan- dem mass spectrometry; FPLC, fast performance liquid chromatography; HexCer, hexosylceramide; HILIC, hydrophilic interaction chromatography; IS, internal standard; LacCer, lactosylceramide; LOD, limit of detection; LPDS, lipoprotein deficient serum; MRM, multiple reaction monitoring; S.D., standard deviation; S1P, sphingosine-1- phosphate; SPC, sphingosylphosphorylcholine; SPH, free sphingoid base; TrimetSPH, trimethyl-sphingosine; var., variable ⁎ Corresponding author. Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, D-93053 Regensburg, Germany. Tel.: + 49 941 944 6240; fax: +49 941 944 6202. E-mail address: gerhard.liebisch@klinik.uni-regensburg.de (G. Liebisch). 1388-1981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2010.11.003 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip