Isolation and characterisation of intact steryl ferulates from seeds

Approaches to the isolation and characterisation of intact steryl ferulates were investigated. Alkaline washing of the total lipid extract obtained from seeds allowed preliminary separation of a phenolic ester containing fraction from the abundant neutral lipids (comprising largely triacylglycerides, sterols and steryl fatty acyl esters). Subsequent thin-layer chromatography (silica gel) afforded purified steryl ferulates. Separation of individual molecular species was achieved by reversed-phase (ODS) high-performace liquid chromatography (HPLC) using the acetate derivatives of the steryl ferulates, or by high-temperature gas chromatography (GC) of the underivatised steryl ferulates or their acetates or trimethylsilyl ethers. Characterisation of intact individual molecular species was based on the electron impact mass spectra obtained by GC—mass spectrometry (MS) of the underivatised ferulates or their trimethylsilyl ethers, or by direct insertion probe MS of the acetates of the steryl ferulates obtained by preparative HPLC. The feasibility of the above approach was established using the well characterised steryl ferulate mixture γ-oryzanol obtained from rice bran oil. The methods were subsequently applied to the analysis of the steryl ferulates present in seeds of maize ( Zea mays ).