Lipids are important natural products and essential in nutrition, cosmetic formulations, pharmaceuticals, etc. Lipids and, particularly, phospholipids are of substantial medical interest (some are molecules with messenger function) and of diagnostic potential (for instance, the lipoproteins in human blood). Among the different soft-ionization mass spectrometric methods that enable detection of the intact lipid molecules, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has several advantages, for instance, simple performance, high sensitivity, and robustness against contaminants. Additionally, MALDI-TOF MS analyzes a solid sample. This enables (in contrast with isotropic solutions) acquisition of spatially-resolved mass spectra (‘mass spectrometric imaging’). However, separation of complex mixtures into the individual lipid classes is normally required to enable detection of all the components. It will be shown with the example of a lipid extract from hens’ egg yolk that MALDI-TOF MS can be easily combined with TLC, enabling detection of as little as picomole amounts of lipids directly on the HPTLC plate. This results in sensitivities higher than those from established staining procedures. Additionally, because of the substantial spatial resolution, lipids separated by normal-phase TLC may not only be differentiated according to differences of their headgroups but also according to differences of their fatty acyl composition. Finally, MS-MS experiments, providing further insights into the structures of the relevant lipids, can be also performed directly on the HPTLC plate. Although the HPTLC-MALDI coupling can be easily established, there are different points to which special attention should be paid. Aspects of matrix application, data acquisition (including the stability of lipids and reproducibility), and data evaluation will be emphasized in this paper
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