A single extraction 96-well method for LC-MS/MS quantification of urinary eicosanoids, steroids and drugs

Urinary eicosanoid concentrations reflect inflammatory processes in multiple diseases and have been used as biomarkers of disease as well as for stratification in precision medicine. However, implementation of urinary eicosanoid profiling in large-scale analyses is restricted due to sample preparation limits. Here we demonstrate a single solid-phase extraction of 300µL urine in 96-well-format for prostaglandins, thromboxanes, isoprostanes, cysteinyl-leukotriene E4 and the linoleic acid-derived dihydroxy-octadecenoic acids (9,10- and 12,13-DiHOME). A simultaneous screening protocol was also developed for cortisol/cortisone and 7 exogenous steroids as well as 3 cyclooxygenase-inhibitors. Satisfactory performance for quantification of eicosanoids with a proper internal standard was demonstrated for intra-plate analyses (CV=8.5-15.1%) as well as for inter-plate (n=35) from multiple studies (CV=22.1-34.9%). Storage stability was evaluated at -20 °C, and polar tetranors evidenced a 50% decrease after 5 months, while the remaining eicosanoids evidenced no significant degradation. All eicosanoids were stable over 3.5-years in urine stored at -80°C. This method will facilitate the implementation of urinary eicosanoid quantification in large scale screening. [Display omitted] •A total of 19 urinary eicosanoids, 9,10- and 12,13-DiHOME were retained by a single SPE extraction•The SPE protocol was simplified to be conducted in 96-well format requiring only 300µL urine•The method can screen for the detection of cortisol/cortisone, 7 exogenous steroids and 3 COX-inhibitors•All 19 urinary eicosanoids were stable over 3.5 years at -80 °C•Average intra-plate CVs were 8.5-15.1% and average inter-plate CVs (n=35) were 22.1-34.9% for eicosanoids and DiHOMEs.