Development of a C18 Supercritical Fluid Chromatography-Tandem Mass Spectrometry Methodology for the Analysis of Very-Long-Chain Polyunsaturated Fatty Acid Lipid Matrices and Its Application to Fish Oil Substitutes Derived from Genetically Modified Oilseeds in the Aquaculture Sector

Abstract

Lipidomics methodologies traditionally utilize either reverse phase-or hydrophilic interaction liquid chromatography type separations; however, supercritical fluid chromatography can offer a rapid normal phase type separation while reducing the dependence on organic solvents. However, normal phase type lipid separations typically lack pronounced intraclass separation, which is problematic for complex lipidomes containing very-long-chain polyunsaturated fatty acids, especially those from genetically modified organisms. A high-strength silica C18 method was developed, which benefitted from discrete class separation, as well as displaying intraclass selectivity sufficient for profiling flesh of salmon fed with a diet supplemented with oil from the genetically engineered oilseed Camelina sativa, a terrestrial oilseed with a fish oil-type profile. Salmon fed a diet containing this Camelina oil were found to have flesh enriched in triacylglycerols and phospholipids containing 18:3, 20:5, and 22:6, whereas salmon fed the control diet were differentiated by shorter chain plant-type fatty acids integrated within complex lipids. Coupled with active scanning quadrupole technology, data acquisition was enhanced, allowing for fragmentation data to be acquired in a data independent fashion, permitting acyl chain identification of resolved isomers. Therefore, we have developed a method, which is amenable for lipidomics studies of complex lipidomes, specifically those altered by synthetic biology approaches. ■ INTRODUCTION The field of lipidomics is one of the most recent omics techniques, evolving from metabolomics, and addressing biological problems, which were not extensively provisioned for by metabolomics, traditionally focused toward the polar end of the metabolite spectrum. Lipidomics has predominantly been carried out using both reverse phase and normal phase hydrophilic interaction liquid chromatography (HILIC), depending on whether separation by the acyl chain or head group was required. Supercritical fluid chromatography (SFC) has been utilized for several decades; however, it is only relatively recently that it has been promoted as a mature analytical technique. SFC is highly amenable to nonpolar compounds, with supercritical CO 2 displaying the polarity of hexane, as well as demonstrating a low viscosity allowing for higher flow rates with small particle columns, resulting in more efficient separations when compared with normal phase chromatography, its closest chromatographic relative. With recent advances in hardware, the ability to consistently reproduce chromatographic behavior is now possible with, for example, the inclusion of automatic back pressure regulators (ABPR) and new pumps designed specifically for supercritical fluids. However, SFC has not seen widespread adoption within the lipidomics field and to date, only a few applications have been described, ranging from the silica-like bridged ethylene hybrid (BEH) column, 1,2 method development across multiple columns, 3,4 and C18 columns. 5,6 Currently, the BEH or similar column is used for lipid class separation, whereas the high-strength silica (HSS) C18 column has been used predominantly for neutral lipids, such as for free fatty acid and triacylglycerol analysis. 7 One disadvantage to using a BEH