Development of a highly sensitive liquid chromatography-mass spectrometry method to quantify plasma leukotriene E₄ and demonstrate pharmacological suppression of endogenous 5-LO pathway activity in man

Low basal endogenous concentrations (<20 pg/mL) of the 5-lipoxygenase (5-LO) pathway biomarker leukotriene E₄ (LTE₄) in human plasma present a significant analytical challenge. Analytical methods including liquid chromatography-mass spectrometry and enzyme linked immunosorbent assays have been used to quantify plasma LTE₄ in the past but have not provided consistent data in the lower pg/mL-range. With our new method, a detection limit (<1 pg/mL plasma) significantly below basal levels of LTE₄ was achieved by combining large volume sample purification and enrichment by anion-exchange mixed mode solid phase extraction (SPE) with large volume injection followed by chromatographic separation by ultra performance liquid chromatography (UPLC) and quantification by highly sensitive negative-ion electrospray tandem mass spectrometry (MS/MS). The method was reproducible, accurate and linear between 1 and 120 pg/mL plasma LTE₄. The method was used to perform an analysis of plasma samples collected from healthy volunteers in a Phase 1 study with the FLAP (5-lipoxygenase activating protein) inhibitor AZD5718. Basal endogenous LTE₄ levels of 5.1 ± 2.7 pg/mL were observed in healthy volunteers (n = 34). In subjects that had been administered a single oral dose of AZD5718, significant suppression (>80%) of plasma LTE₄ level was observed, providing pharmacological evidence that endogenous 5-LO pathway activity could be assessed.