Mass spectrometric methods for analysis of sterols and steryl esters in biological samples

Hailat, Iyad Ali (2014) Mass spectrometric methods for analysis of sterols and steryl esters in biological samples. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The composition of free sterols in cultivated blue mussels obtained commercially and from laboratory feeding experiments has been investigated. Total lipids were extracted from mussels using a modified Folch procedure followed by isolation of free sterols using column chromatography. Sterols were identified using GC/MS and quantified using (GC/MS-SIM) and GC/FID. The most abundant free sterols in commercially cultivated mussels were cholesterol, desmosterol, and brassicasterol. Mussels were fed on algae or fish waste for six months. The concentrations of sterols in fish waste-fed mussels were compared with those in algae-fed mussels. However, fish waste-fed mussels had significantly higher cholesterol concentrations compared to algae-fed mussels. Mussels fed algae had significantly higher campesterol compared to fish waste-fed mussels. Free sterols are neutral molecules that are difficult to ionize by MALDI or ESI. Therefore, in order to increase their ionization efficiency, sterols were converted into their corresponding picolinyl esters, N-methylpyridyl ethers and sulphated esters. Using MALDI-MS, sterol picolinyl esters were identified as [M+Na]⁺, the signal being significantly enhanced after the addition of sodium acetate. Sterol N-methylpyridyl ethers were detected as [M]⁺ while sulphated sterols were detected as [M-H]⁻. The ester bonds of picolinyl and sulphated esters cleaved during MALDI-CID MS/MS resulting in diagnostic fragments at m/z 146 and 97, respectively. Picolinyl esters of sterols were also analyzed using ESI/APCI-QIT MSn. The Picolinyl esters were detected as [M+H]⁺ in ESI and APCI sources. The ester bonds of picolinyl esters cleaved during CID MS², resulting in diagnostic fragments corresponding to steryl cation moieties [M+H-C6H5NO2]⁺. The CID MS³ of [M+H]⁺ → [M+H-C6H5NO2]⁺ of picolinyl esters was found to be useful for structural elucidation and for distinguishing among steryl isomers. Steryl esters were identified in margarine and corn using ESI-QIT MSn. Sterols and other lipids extracted from samples using hexane were subjected to solid phase extraction. The steryl ester fraction was eluted with hexane: diethyl ether (98:2, v/v). Fatty acid steryl esters were detected as [M+NH4]⁺ using ammonium acetate as dopant. Steryl esters, including molecular isomers, were identified using ESI-QIT MS². ESI-QIT MS³ was carried out on the intact steryl fragmentation cation. The resulting CID spectra of the steryl cation were found to be unique and similar to those from APCI-QIT MS² CID of free sterol standards.

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