Strategy for the analysis of steryl esters from plant and animal tissues

Methods are described for the analysis of intact steryl esters present in complex mixtures isolated from plant or animal tissues. A preliminary examination by analytical thin-layer chromatography (TLC) and capillary column gas chromatography-mass spectrometry (GC-MS) under electron impact (EI) ionisation reveals the complexity of the mixture and the nature of the steryl moieties. Preparative TLC is then utilised to separate the steryl esters into two broad groups, containing fatty acyl moieties of shorter (C2-C8) or longer chain length (C10-C22). The shorter-chain fatty acyl steryl esters are separated by adsorption high-performance liquid chromatography (HPLC) on a LiChrosorb Silica-60 column. The steryl esters with longer-chain fatty acyl moieties are analysed by reversed-phase HPLC on either an Ultrasphere ODS, 5-micron, or a S3 Spherisorb ODS, 3-micron, column. Steryl esters with unsaturated fatty acyl moieties are eluted with the shorter-chain fatty acyl steryl esters. The presence of the unsaturated fatty acyl esters can be monitored by analytical argentation TLC, which will also reveal the degree of unsaturation. The steryl esters are fractionated into the saturated, mono-, di-, tri- and polyene acyl types by preparative medium-pressure liquid chromatography on a column of 10% AgNO3-silica gel. Each of these steryl ester types can then be resubmitted to reversed-phase HPLC or analysed by GC-MS on a short fused-silica capillary column with a bonded phase of the OV-1 type. GC-MS on a magnetic-sector instrument under negative-ion chemical ionisation conditions with ammonia as the reagent gas produces fragment ions for both the steryl and fatty acyl moieties, thus permitting identification of the individual intact steryl esters. These various methods are illustrated by analyses of the steryl ester mixtures obtained from human plasma, barley seedlings, palm oil and rape seed oil.