Deducing formation routes of oxylipins by quantitative multiple heart-cutting achiral-chiral 2D-LC-MS

This work describes the first two-dimensional (2D) liquid chromatography (LC) method that combines comprehensive quantitative analysis of oxylipins with chiral separation. By means of multiple heart-cutting (MHC), the peaks of the oxylipins are collected from a 31.5 min efficient reversed-phase (RP) 1D-separation and transferred onto a short chiral column with < 2 µm particles. Full gradient 2D separation of 45 pairs of enantiomeric hydroxy- and vicinal dihydroxy-fatty acids is achieved within 1.80 min, resulting in peak widths at half height < 2.5 sec. Precise heart-cutting of the complete 1D peak enables high sensitivity in tandem-mass spectrometric detection following electrospray-ionization (ESI) in negative mode, yielding with lower limits of quantification below 1 pg on column. Enantiomeric fractions of oxylipins can be precisely (±5%) determined even at low concentrations or high enantiomeric excess of one isomer. Combined with comprehensive quantification in 1D both, the concentration as well as the enantiomeric ratio can be determined in biological samples. This is demonstrated for hydroxy-fatty acids in cell culture, enabling to distinguish formation by (acetylated) cyclooxygenase-2 as well as radical mediated autoxidation. Analysis of 5,15-DiHETE, 5,15-DiHEPE and 7,17-DiHDHA, whose four stereoisomers coelute in RP-LC, showed that in human M2-like macrophages the (S,S)-enantiomer is predominantly present, indicating enzymatic formation. The achiral-chiral MHC-2D-LC-ESI(-)-MS/MS method thus improves the targeted oxylipin analysis and offers unprecedented selectivity, enabling an understanding of the formation route of these lipid mediators.