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

Introduction: Several oxylipins are key regulators of inflammation. Those are formed by enzymes such as lipoxygenases or cyclooxygenases but also by autoxidation. While the autoxidation is stereorandom, only individual stereoisomers are formed enzymatically. Oxylipin formation is comprehensively analyzed by reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, simultaneously quantifying > 150 oxylipins including numerous positional isomers. Enantiomers are not separated and enantioselective methods lack sensitivity and selectivity. Objectives: Development and application of a method enabling the sensitive, specific and enantioselective analysis of oxylipins allowing the investigation of the formation routes of hydroxy- and dihydroxy-fatty acids in biological samples. Methods: An achiral-chiral multiple heart-cutting two-dimensional (2D)-LC-MS/MS method was developed. The separation of 45 pairs of enantiomeric hydroxy- and vicinal dihydroxy-fatty acids is achieved within 1.80 min 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. The method was applied on the analysis of oxylipins in human cells. Results: In human M2-like macrophages, the so-called specialized pro-resolving mediators (SPM) 5,15-DiHEPE and 7,17-DiHDHA as well as 5,15-DiHETE occurred as (S,S)-enantiomer, supporting their enzymatic formation. In contrast, at least eight isomers of 10,17-DiHDHA are present in immune cells indicating formation by autoxidation. Consistent with the literature, in human plasma of healthy subjects these dihydroxy-fatty acids are not present. However, they quickly form by autoxidation if the samples are stored improperly, yielding all four stereoisomers. Conclusion: Using the first two-dimensional liquid chromatography method for the quantitative analysis of oxylipins with chiral separation, we could show that not only the specific enantiomers of dihydroxy- PUFA described as SPM are present in biological samples but numerous isomers can be found. These are rapidly formed during inappropriate sample storage. Thus, dihydroxy-FA should only be reported as SPM if an enantioselective analysis has been carried out.