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 quantitative analysis of oxylipins with chiral separation. By multiple heart-cutting (MHC), the peaks of the oxylipins are collected from a 31.5 min reversed-phase (RP) 1 D-separation and transferred onto a short chiral column with < 2 µm particles. Full gradient 2 D 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. Heart-cutting of the complete 1 D peak enables high sensitivity in tandem-mass spectrometric detection following electrospray-ionization (ESI) in negative mode, yielding 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. These dihydroxy-fatty acids are not detected in plasma from healthy subjects. However, they quickly form by autoxidation if the samples are stored improperly, yielding all four stereoisomers. 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.