Analysis of Free and Esterified Sterol Content and Composition in Seeds Using GC and ESI-MS/MS

Total sterol content and composition in plant tissues can be easily determined by gas chromatography (GC) after saponification of the total lipid extract. However, in oleogenic tissues a significant proportion of the sterol is esterified to fatty acids, with GC methodologies unable to provide information about the proportion and the molecular species composition of intact steryl esters (SEs). Here we describe an electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and Multiple Reaction Monitoring (MRM) method which, in parallel with GC analysis, allows for the accurate determination of both free and esterified sterol content and composition in seeds. After extraction of seed oil with hexane, free sterols are derivatized with undecanoyl chloride, total steryl esters are then purified from triacylglycerol (TAG) by liquid chromatography, infused and ionized as ammonium adducts, with molecular species identified and quantified by fragmentation in the presence of internal standards.