Tetra-acylated sucrose esters (SE) in Turkish tobacco extracts have been separated and identified using liquid chromatography-mass spectrometry (LC-MS). An enriched fraction of SE from semi-preparative normal phase liquid chromatography of the methylene chloride extract was chosen for characterization. Several types of SE were observed that differed primarily in placement of the acyl functionality on either the glucose or the fructose fragment. In addition to acetyl, the other three observed acyl substitution patterns were governed by the presence of butyl, pentyl, and hexyl alkyl substituents. Within each type of SE, there were observed several homologs differing by a methylene moiety. Within each homolog, a number of isomeric components were found. The presence of unsaturation in one of the acyl alkyl substituents gave rise to another homologous series wherein isomers of the same mass were discovered. The information on the substitution pattern of SE using this new LC-MS approach was very similar to that obtained previously on tobacco samples employing gas chromatography-mass spectrometry (GC-MS) of derivatized sucrose esters.