Column coupling for chiral LC-MS separation resolves triacylglycerol enantiomers and regioisomers in complex samples...

The positional distribution of fatty acids in the glycerol of triacylglycerols (TGs) gives multiple regioisomeric and enantiomeric species with different absorption, metabolism and tissue distribution. This diversity is further enhanced by the positional isomers of double bonds in these fatty acids which present an analytical challenge. To improve the determination of this plethora of isomers, we coupled chiral liquid chromatography with electrospray ionization high-resolution mass spectrometry. For the separation of enantiomers and regioisomers, we used an amylose tris(3-chloro-4-methylphenylcarbamate) stationary phase (Chiralpak IF-3) and evaluated the key chromatographic parameters: mobile phase composition, column temperature and flow-rate. Acetonitrile/water (98:2, v/v) as mobile phase at 35 °C and 0.15 mL/min was selected for separation of enantiomers and regioisomers. In addition, coupling three columns (750 mm) improved efficiency and resolution for the separation of key enantiomers and regioisomers. In addition, our method discriminated TGs differing in double-bond position and cis/trans configuration, which are difficult to resolve using conventional reversed-phase chromatographic systems. The separation method showed stable retention and reproducible determination of enantiomeric percentages in liver matrices. The application of the method revealed the stereochemical TG diversity in rat liver extracts that is unresolved by conventional RP chromatography. Furthermore, it enabled detection of differences in enantiomeric distribution following exposure to doxorubicin. This highlights the importance of resolving TG isomers in biological samples. In conclusion, this work reports a robust MS-compatible lipidomics chiral platform for stereospecific TG analysis providing an analytical foundation for future investigation of lipid remodelling and de novo lipogenesis at the stereochemical level.