UHPLC-MS/MS method for chiral separation of 3-hydroxy fatty acids on amylose-based chiral stationary phase and its application for the enantioselective analysis in plasma and platelets.

3-Hydroxyfatty acids (3-OH-FAs) are formed in the hydration step during mitochondrial β-oxidation of saturated straight-chain fatty acids, which is a catabolic pathway that involves several enzymes. For an unbiased biological interpretation, an enantioselective analysis of 3-OH-FAs including their stereoisomers is necessary, which may contribute to the elucidation of enzymatic mechanisms in the biological pathways. In this work, an enantioselective gradient UHPLC-MS/MS method based on 1.6 µm particle polysaccharide column (Chiralpak IA-U) for chiral separation of 3-hydroxyfatty acids was developed which covers carbon chain length from C8 to C18 with a good resolution of R and S enantiomers. The method is fast and sensitive for detecting enantiomers of 3-OH-FAs by using a triple quadrupole instrument as a detector in a targeted, selected reaction monitoring (SRM) mode. A matrix matched-calibration strategy was applied for quantification of individual 3-OH-FA enantiomers. The method allows the simultaneous quantification of each enantiomer of 3-OH-FAs from C8-C18. One-phase liquid extraction with 2-propanol showed good extraction recoveries with over 90% on average. Further, the validated method was applied to investigate the alteration of 3-OH-FA enantiomers in platelets and plasma samples from human donors with different diagnoses of cardiovascular disease (acute coronary syndrome ACS, chronic coronary syndrome CCS). Both R and S enantiomers were detected in platelets and plasma samples with different predominance for R or S in dependence on carbon chain length, which might be associated with different functional enzymes of mitochondrial and peroxisomal β-oxidation. Finally, our study provides a new strategy for chiral separation and enantioselective analysis, showing great potential for targeted metabolomics in clinical biomarker discovery.