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A blotting method that transfers analytes separated on wettable high-performance thin-layer chromatography (HPTLC) plates to a hydrophobic reversed-phase C8 HPLTC plate suitable for analysis with a liquid microjunction surface sampling probe electrospray ionization mass spectrometry system was described and demonstrated. The simple blotting procedure transfers the analyte from the wettable plate to the topmost surface of a rigidly backed, easy-to-mount hydrophobic substrate that already has been proven viable for analysis by this sampling probe/mass spectrometry system. The utility of the approach was demonstrated by the analysis of a four-component peptide mixture originally separated on a ProteoChrom® HPTLC cellulose sheet and then blotted onto the reversed-phase HPTLC plate. Published in 2011 by John Wiley & Sons, Ltd.