Characterization And Quantification Of Triacylglycerols In Edible Oils By Two-dimensional Liquid Chromatography Using A Single Column Coupled With Mass Spectrometry

Triacylglycerols (TAGs) are the main component of plant oils (95%98%). The physicochemicaland nutritional properties of oils are determined by TAG molecules, thus, TAGs in edible oils areconsidered as good fingerprints for authenticity control. The profiling analysis of TAGs in edible oils isa challenging task because of the presence of numerous TAGs species with similar physicochemicalproperties.Non-aqueous reversed-phase HPLC (NARP-HPLC) and silver ion HPLC (Ag-HPLC) are the twomain methods for the separation of complex TAGs in natural samples. In NARP-HPLC, the retention ofTAGs is governed by the equivalent carbon number (ECN). However, NARP system has a lowerselectivity for the separation of TAGs with the same ECN and TAG regioisomers. The complementaryseparation mode, Ag-HPLC, can separate TAGs with the same ECN and TAG regioisomers. Theresolution and peak capacity of off-line or on-line two-dimensional (2D) LC system coupling NARPand Ag-HPLC have been improved tremendously. However, off-line approaches are generallytime-consuming and laborious; Furthermore, it requires a large amount of organic solvents, which maysubsequently lead to additional environmental pollution. Some advantages of on-line2D methodscompared with off-line ones are the fast speed and automation; on the other hand, some drawbacks arethe complexity of the system and the incompatibility of the2D solvents. Therefore, it is urgent todevelop an efficient and convenient chromatographic method for separation TAGs in edible oils.This project focus on the development of a new method for the efficient separation and analysis ofTAGs, which is the main constituent of oils and fats, and solve the major technical bottleneck on edibleoil safety research. The stationary phase in phenyl-hexyl column can be considered as a mixed-modestationary phase, in which phenyl moiety could provide π-π interactions with the π electrons of DBs andthe hexyl ligands could provide additional hydrophobic interactions. Chromatographic behaviors ofphenyl-hexyl column were investigated, and separation conditions were optimized. We developed an2DHPLC system using a single column coupled with atmospheric pressure chemical ionization (APCI)mass spectrometry, and an quantization method by using response factors approach. Then this systemwas applied for identification and quantification of TAGs in plant oils. The major results are as follows:1. Chromatographic behaviors of phenyl-hexyl column were investigated. Phenyl-hexyl columncan provide both hydrophobic interactions for the separation of TAGs with different ECNs as well asπ-π interactions for the separation of TAGs with different DBs. This column exhibited much higherselectivity and excellent stability.2. A method for online separation of triacylglycerols (TAGs) in plant oils by two-dimensional (2D)liquid chromatography using a single column coupled with APCI mass spectrometry was developed.The2D LC-MS system was applied for the profiling of TAGs in soybean oils, peanut oils, corn oils, andsesame oils.29TAGs in peanut oils,22TAGs in soybean oils,19TAGs in corn oils, and19TAGs insesame oils were determined. The LC-MS data was analyzed by barcodes and principal component analysis (PCA). The resulting barcodes and PCA enabled a clear identification of different plant oils.The simple, rapid and efficient method provides an efficient and convenient chromatographictechnology for the fast characterization and quantification of complex TAGs in plant oils and otherlipids.3. The off-line2D HPLC system using a single column coupled with APCI mass spectrometry hasbeen developed for the identification and quantification of TAGs. The method was successful in theseparation of a higher number of TAG solutes. In addition, TAG quantization is achieved by usingresponse factors approach with APCI/MS to improve accuracy. Then this system was applied for theprofiling of soybean oil, peanut oil and lord oil, totally68TAGs including40TAGs in soybean oils,50TAGs in peanut oils and44TAGs in lord oils were identified and quantified. The LC-MS data wasanalyzed by PCA. The statistical multivariate data analysis enables a clear identification of adulteratedsoybean oils from5%of added lord oil and peanut oils from5%of added soybean oil as an adulterant.This approach is simple and practical, which can identify more TAGs and quantify TAGs more reliable.It offers a potential application for edible oil authenticity control and adulteration detection.