Development of Liquid Chromatographic and Mass Spectrometric Methods for the Identification of Chlorinated Triacylglycerols in Natural Oils

Abstract

ABSTRACT

There is a growing concern about halogenated organic compounds, in particular organochlorines, entering the food chain and causing adverse health effects to humans. Known persistent halogenated anthropogenic substances, such as PCBs and DDT, account for only 10-15% of the total. The remaining 85-90% may contain chlorinated triacylglycerols (Cl-TAGs) which have been investigated to some extent using gas chromatography (GC). Progress in this research area is rather limited due to the lack of appropriate reference samples and reliable methods. The main objective of this thesis project was to develop analytical methods for the characterization of Cl-TAGs in natural oils, particularly corn and salmon oils. Since no certified Cl-TAGs were commercially available, our attention was first focused on their synthesis. Four new Cl-TAGs were synthesized using enzyme-catalyzed and carbodiimide-mediated coupling reactions. They were then characterized by ESI(+)/MS and NMR techniques. An untreated commercially available corn oil sample was studied extensively and 25 natural TAGs were identified in it by eight methods developed in this thesis. These methods are: GC/MS, both off-line and on-line 2D Ag-HPLC and RP-HPLC, off-line 2D RP-HPLC/GC/MS, off-line 2D Ag-HPLC/ESI(+)/MS, off-line 2D RP-HPLC/ESI(+)/MS, and a new off-line 3-D Ag-HPLC/ESI(+)/MS/GC/MS. In addition, 10 oxidized TAGs were identified in the corn oil sample by an off-line RP-HPLC/ESI(+)/MS method. These oxidized TAGs have not yet been reported in the literature. The corn oil was then selected as a representative oil. It was spiked with four Cl-TAGs synthesized in this thesis and then studied using the off-line RP-HPLC/ESI(+)/MS method. Among the eight methods developed to study corn oil only two, namely GC/MS and RP-HPLC/ESI(+)/MS, were applied to characterize TAGs in raw farmed salmon skin and tissue oils. The off-line RP-HPLC/ESI(+)/MS method was successfully used to identify 88 TAGs in the salmon skin oil and 81 TAGs in the salmon tissue oil. This is first time TAGs in raw farmed salmon skin and tissue oils have been directly identified. Attempts were also made to identify Cl-TAGs in the salmon oil. Only four TAGs, so far unknown, were identified using the off-line RP-HPLC/ESI(+)/MS method. Doubly unsaturated fatty acids of C16 and C18 (e.g., 3 and 4) were detected in the CID spectra but not by GC/MS. No information on these fatty acids is available in the literature.