Isolation of lipid classes from nutritional marine oils: improved extraction strategy and discrimination studies based on the positional distribution of omega-3 polyunsaturated fatty acids on triacylglycerol structures using liquid chromatography tandem mass spectrometry and principal component analysis

Abstract

The consumer of the 21st century have developed an awareness of the quality of food products in their diet and the impact of these products on their health which in turn has led to an escalating consumer demand for omega-3 fatty acids rich oils, functional food, dietary supplements and pharmaceuticals. The analysis of omega-3 rich oils for detecting the presence of adulterants is generally carried out by using complex, time-consuming techniques and expensive and sophisticated instruments. It is therefore essential to establish simple and reliable analytical methods in order to carry out quality assessment and authentication of nutritional omega-3 fatty acids rich products. The most critical factors clearly affecting the analysis of lipids in a wide variety of samples are the extraction and isolation steps due to the presence of various lipid classes, which in turn demand the pre-separation of the sample prior to fatty acid methyl ester (FAME) compositional analysis by gas chromatography (GC) or liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS2). In this work, a simple, rapid, and cost effective novel liquid-liquid extraction (LLE) method is developed to separate triacylglycerol (TAG) and phospholipids (PL) from marine oils. Krill oil was selected as analytical sample due to its content of TAG and phospholipids.The method consists basically of sequentially adding methanol and hexane to the oil sample, separating and washing the phases with solvents of opposite polarities. The result revealed that it is possible to separate the major TAG and PL constituents from krill oil samples. The high performance thin layer chromatography (HPTLC) chromatograms revealed that TAG and PL were absent from the PL and TAG rich fraction respectively. The thesis also studies the capability of stereospecific positioning for discriminating marine oils based on their number of omega-3 polyunsaturated fatty acids (omega-3 PUFAs) at sn-2 position of TAG structures. The fatty acid distribution on TAG molecules was determined by using LC-ESI-MS2 and a previously developed algorithm. The results were arranged in increasing number of equivalent carbon number (ECN) and the omega-3 PUFAs at sn-2 positions were counted and presented in a histogram. The results revealed that discrimination studies based on the sn-2 position are reasonable alternatives for discriminating genuine from processed marine oils. The final aspect studied in the present thesis is the implementation of a new strategy for discriminating marine oils by using the position of omega-3 PUFAs at sn-1, sn-2 and sn-3 combined with principal component analysis (PCA). The novel strategy demonstrated to be a reliable approach to discriminate not only genuine but also processed and intentionally adulterated oils.