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

Prostaglandins Other Lipid Mediat. 2020 Oct:150:106463. doi: 10.1016/j.prostaglandins.2020.106463. Epub 2020 May 23.

Abstract

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.

Keywords: 5-lipoxygenase; AZD5718; Cardiovascular disease; FLAP; LTE4; Leukotriene E4; Liquid chromatography; Mass spectrometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arachidonate 5-Lipoxygenase / metabolism*
  • Biomarkers / blood
  • Chromatography, Liquid / methods*
  • Clinical Trials, Phase I as Topic
  • Humans
  • Leukotriene E4 / blood*
  • Lipoxygenase Inhibitors / administration & dosage
  • Pyrazoles / administration & dosage*
  • Randomized Controlled Trials as Topic
  • Tandem Mass Spectrometry / methods*

Substances

  • AZD5718
  • Biomarkers
  • Lipoxygenase Inhibitors
  • Pyrazoles
  • Leukotriene E4
  • Arachidonate 5-Lipoxygenase