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Lipidomics is a rapidly expanding area of scientific research and there are a number of analytical techniques that are employed to facilitate investigations. One such technique is matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Previous MALDI-MS studies involving lipidomic investigation have included the analysis of a number of different ex vivo tissues, most of which were obtained from animal models, with only a few being of human origin. In this study, we describe the use of MALDI-MS, MS/MS and MS imaging methods for analysing lipids within cross-sections of ex vivo human skin. It has been possible to tentatively identify lipid species via accurate mass measurement MALDI-MS and also to confirm the identity of a number of these species via MALDI-MS/MS, in experiments carried out directly on tissue. The main lipid species detected include glycerophospholipids and sphingolipids. MALDI images have been generated at a spatial resolution of 150 and 30 μm, using a MALDI quadrupole time-of-flight Q-Star Pulsar- i (Applied Biosystems/MDS Sciex, Concord, ON, Canada) and a MALDI high-definition MS (HDMS) SYNAPT G2-HDMS system (Waters, Manchester, UK), respectively. These images show the normal distribution of lipids within human skin, which will provide the basis for assessing alterations in lipid profiles linked to specific skin conditions e.g. sensitisation, in future investigations. [ABSTRACT FROM AUTHOR]