Background: Lipids such as phosphatidic acids (PAs) and cardiolipins (CLs) present strongly tailing peaks in reversed phase liquid chromatography, which entails low detectability. They are usually analyzed by hydrophilic interaction liquid chromatography (HILIC), which hampers high-throughput lipidomics. Thus, there is a great need for improved analytical methods in order to obtain a broader coverage of the lipidome in a single chromatographic method. We investigated the effect of ammonium bicarbonate (ABC) on peak asymmetry and detectability, in comparison with ammonium formate (AFO) on both a conventional BEH C18 column and an HST-CSH C18 column.

Results: The combination of 2.5 mM ABC buffer pH 8 with an HST-CSH C18 column produced significantly improved results, reducing the asymmetry factor at 10 % peak height of PA 16:0/18:1 from 8.4 to 1.6. Furthermore, on average, there was up to a 54-fold enhancement in the peak height of its [M - H]- ion compared to AFO and the BEH C18 column. We confirmed this beneficial effect on other strongly tailing lipids, with accessible phosphate moieties e.g., cardiolipins, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, phosphorylated ceramide and phosphorylated sphingosine. Furthermore, we found an increased detectability of phospho- and sphingolipids up to 28 times in negative mode when using an HST-CSH C18 column. The method was successfully applied to mouse liver samples, where previously undetected endogenous phospholipids could be analyzed with improved chromatographic separation.

Significance: In conclusion, the use of 2.5 mM ABC substantially improved the peak shape of PAs and enhanced the detectability of the lipidome in negative mode on an RPLC-ESI-Q-TOF-MS system on both BEH C18 and HSTCSH C18 columns. This method provides a wider coverage of the lipidome with one single injection for future lipidomic applications in negative mode.

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.