Simultaneous Determination of Phytosterol Conjugates in Brown Rices and Legumes
Date Issued
2012
Date
2012
Author(s)
Su, Ding-Yuan
Abstract
There are five common forms of phytosterols: free sterols (FS), steryl ferulates (SF), steryl glycosides (SG), acylated steryl glycosides (ASG) and steryl esters (SE). Traditional phytosterol analysis methods have many drawbacks, for instance, acid hydrolysis may cause sterol degradation or isomerization. The aim of this study is to develop methods to determine free and conjugated phytosterols simultaneously by LC/APCI-MS (liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry) and LC-ELSD (liquid chromatography- evaporative light scattering detector). Phytosterols could be separated by NH2 SPE cartridge while triglycerides and steryl esters were removed from sample during sample clean-up. Recoveries of added free sterols analyzed by LC/APCI-MS were 83.69 to 106.89 %. Replacing hydrolysis methods by solid phase extraction could avoid sterol isomerization and retain intact sterol conjugates. Single compounds of FS, SF, SG and ASG were separated by reverse phase C18 column and detected by APCI-MS simultaneously. All phytosterols give an intense characteristic ion corresponding to the loss of a water molecule from the sterol moiety. The limit of detection (LOD) for free sterols was 5-50 ng/mL. NPLC-ELSD could separate and detect lipid (phytosterol) classes synchronously without further sample pretreatment. Sterol esters still could not be quantified by NPLC-ELSD because of the coelution of wax esters and steryl esters. The limit of detection (LOD) for phytosterols was 0.5-5 μg/mL. Results showed significant difference in the phytosterol contents of samples between measurement by LC-MS and LC-ELSD. Since the lack of single pure standard of phytosterol conjugates, it is hard to quantify accurately by LC-MS. ELSD provides a more uniform response to structurally similar analytes, so here we speculated that the results measured by LC-ELSD were much accurate than LC-MS. Avenasterol, campesterol, stigmasterol and β-sitosterol are predominate among sample phytosterols. The major phytosterols in brown rices are FS and SF (γ-oryzanol) and there is less SG and ASG in brown rices. Legumes all have high content of FS and ASG but no SF. Soybean and black soybean contain much higher amounts of SG than other samples.
Subjects
phytosterol conjugates
brown rice
legume
liquid chromatograph/tandem mass spectrometry
atmospheric pressure chemical ionization
evaporative light scattering detector
Type
thesis
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