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.

人血浆中5-脂氧合酶 (5-LO) 途径生物标志物白三烯 E ₄ (LTE ₄ ) 的低基础内源浓度 (<20 pg/mL) 是一项重大的分析挑战。包括液相色谱-质谱法和酶联免疫吸附测定在内的分析方法过去曾用于量化血浆 LTE ₄，但没有提供较低 pg/mL 范围内的一致数据。使用我们的新方法，检测限（<1 pg/mL 血浆）显着低于 LTE _{4 的}基础水平通过将阴离子交换混合模式固相萃取 (SPE) 的大体积样品纯化和富集与大体积进样、超高效液相色谱 (UPLC) 色谱分离和高灵敏度负离子电喷雾串联质谱定量相结合来实现(MS/MS)。该方法在 1 到 120 pg/mL 血浆 LTE ₄之间具有重现性、准确度和线性。在使用 FLAP（5-脂肪氧化酶激活蛋白）抑制剂 AZD5718 的 1 期研究中，该方法用于对从健康志愿者收集的血浆样本进行分析。基础内源性LTE ₄在健康志愿者（n = 34）中观察到 5.1 ± 2.7 pg/mL 的水平。在单次口服 AZD5718 的受试者中，观察到血浆 LTE ₄水平显着抑制（> 80%），提供了可以评估内源性 5-LO 通路活性的药理学证据。