(T1430-05-29) Chiral Separation of Triacyclglycerols Isomers by Supercritical Fluid Chromatography

Purpose: Triacylglycerols (TAGs), which are the main constituents of natural oils and fats, compose of three fatty acids and glycerol linked with ester bonds. TAGs have various isomers with differing physical and metabolic pathways. There is an established relationship between enantiomer and stereospecific variation of TAGs and digestive uptake in the human gut. As lipid-based delivery and lipid-uptake dependent therapeutics expand, greater knowledge of separation techniques to characterize TAG isomers and enantiomers can benefit future ADME testing as well as formulation development. In this study, we optimized the analytical conditions for TAG isomer separation by SFC using automated method development software in conjunction with triple quadrupole mass spectrometry (TQ-MS) detection.

Methods: TAGs have two different types of isomers. Positional isomers are the TAGs where different fatty acids are connected to the sn-2 position. Enantiomers are the isomers that have chiral centers at the sn-2 position.(Fig.1) These isomers require chromatographic separation due to the challenge of MS separation of isomers with identical molecular weights. To resolve these various isomers on-column, the Daicel CHIRALPAK IG-3 (250×4.6 mm I.D., 5 µm) was selected. Method Scouting Solution software was used to create 27 isocratic analytical conditions with three different oven temperatures, modifier concentrations, and back pressure settings that were investigated. We analyzed standard solution mixtures that contain six type of TAGs isomers, namely: (sn-POO, sn-OOP, sn-OPO, sn-PPO, sn-OPP, sn-POP). See Table 1. for additional detail on SFC and MS method parameters.

Results: By varying the column temperature from 20 C to 40 C, almost no change in selectivity or resolution of the TAG samples was observed. As expected, elution times were moved earlier, and peak widths decreased for higher temperatures. Modifier concentration had a greater effect on the selectivity of separation than temperature with sn-POO and sn-OPP at 40% modifier nearly being resolved, however that modest gain is offset by the lack of increased resolution in the rest of the components. Back pressure was seen to have the greatest effect, providing increased resolution of sn-POO and sn-OPP, as well as other components. In this analysis, the optimal analytical conditions were determined to be: Oven temp. 30 ̊C, Modifier 20%, Back pressure 20 MPa.

Conclusion: We developed an analytical method for TAG isomers using a SFC-MS method development system capable of rapid deployment and automated operation for screening not only mobile phases and stationary phases, but method parameters that can have a key role in optimizing separation for SFC, such as Back Pressure Regulator settings. While TQ-MS was chosen for detection, the methods demonstrated can be adapted for UV or ELSD use.

References: Karupaiah T, Sundram K. Effects of stereospecific positioning of fatty acids in triacylglycerol structures in native and randomized fats: a review of their nutritional implications. Nutr Metab (Lond). 2007 Jul 12;4:16. doi: 10.1186/1743-7075-4-16. PMID: 17625019; PMCID: PMC1947992.

Two types of triacylglyceride isomers


Chromatograms comparing the effect of each analytical parameter