On-Line Overpressure Thin-Layer Chromatographic Separation and Electrospray Mass Spectrometric Detection of Glycolipids

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Abstract
On-line thin-layer chromatographic separation and electrospray mass spectrometry (TLC/ESI-MS) has been accomplished by direct linking of a commercial overpressure TLC instrument, OPLC 50, and a Q-TOF mass spectrometer. Mass spectrometric detection sensitivity and chromatographic resolution achieved by this configuration were assessed using acidic glycolipids as examples. Under the optimized conditions, a sensitivity of 5 pmol of glycosphingolipid was readily demonstrated for TLC/ESI-MS and 20 pmol for TLC/ESI-MS/MS product-ion scanning to derive the saccharide sequence and long chain base/fatty acid composition of the ceramide. Initial preconditioning of TLC plates is necessary to achieve high sensitivity detection by reducing chemical background noise. Plates can be used repeatedly (at least 10 times) for analysis, although this may result in a minor reduction in TLC resolution. Following solvent development, separated components on the TLC plates can be detected in the conventional way by nondestructive staining or UV absorption or fluorescence and can be stored for on-line TLC/ESI-MS analysis at a later stage without reduction in mass spectrometric detection sensitivity and chromatographic resolution. Aspects for further improvement of OPLC instrumentation include use of narrower TLC plate dimensions and refined design of the eluate exit system.