New method to determine free sterols/oxysterols in food matrices using gas chromatography and ion trap mass spectrometry (GC-IT-MS).

Sterols/oxysterols in food may be free or bound i.e. esterified with fatty acids. Methods commonly applied to determine those compounds in such matrices (based on various analytical techniques) usually start with hydrolysis of the food lipid fraction, which means that the results are no good indication of concentration of free sterols/oxysterols only. But only free oxysterols are proatherogenic factors, bound ones are not. There are some published methods selectively sensitive to free oxysterols only, but they are capable to determine only a few compounds and feature very low recovery rates. The aim of this work was to develop a method to determine various free (non-esterified) sterols/oxysterols in various food matrices. The developed method is based on the GC-IT-MS technique used in the chemical ionization mode. It was applied to determine 16 different free sterols/oxysterols in egg powder, cheese, butter, milk and salami. Fat extracted from the given matrix is purified on a specially prepared silica-gel bed to separate the sterol fraction from the oxysterol one. Sterols are silylated using N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane BSTFA:TMCS, then GC-IT-MS analysed. The method features high recovery rates (75-95%), high reproducibility (RSD<20%), and sensitivity within the 0.01-0.3 mg 100 g(-1) range, depending on the analysed compound. The method is ideally suited for determination of free sterols/oxysterols. Besides, should total concentration of both free and bound forms be of interest, food lipids may be transesterificated before the silica-gel bed purification step.