Integrated identification/confirmatory and targeted analysis of epoxyeicosatrienosic acids in human serum by LC-TOF MS and automated on-line SPE-LC-QqQ MS/MS.

A combined strategy is here proposed for qualitative/quantitative targeted analysis of epoxyeicosatrienoic acids (EETs) in human serum. Identification of EET regioisomers was initially carried out by LC-TOF MS in high accuracy mode under optimum conditions for chromatographic separation of the four isomers with an isocratic method using 40:40:20 (v/v/v) methanol-acetonitrile-water containing 0.02% acetic acid. Confirmatory analysis was supported on MS/MS experiments using the hybrid QqTOF mass analyzer by targeted fragmentation of the precursor ion fitting with the molecular formula C₂₀H₃₂O₃ (319.2279 m/z). Identification of selective fragment ions in high accuracy mode enabled the localization of the epoxy functional group and, therefore, the assignation of chromatographic peaks to each EET isomer. After qualitative analysis, an automated method was developed for analysis of EETs in human serum by direct analysis using an on-line platform based on SPE-LC-QqQ MS/MS in selected reaction monitoring. Recovery factors estimated with a dual-cartridge configuration were above 87% for all metabolites either using non-spiked and spiked serum at three different concentrations. Precision, calculated as within-laboratory repeatability and expressed as relative standard deviation, ranged from 2.5 to 9.9% with detection limits below 0.15 ng mL(-1). The optimization of the method was completed with a stability study under different conditions to assess the suited conditions for analysis of EET intermediate metabolites. Finally, concentration ranges of EETs were measured in nine healthy individuals.