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 suggested for patient 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 300 µL 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 an appropriate 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.