Abstract
Eicosanoids are key mediators and regulators of inflammation and oxidative stress often used as biomarkers for diseases and pathological conditions such as cardiovascular and pulmonary diseases and cancer. Analytically, comprehensive and robust quantification of different eicosanoid species in a multi-method approach is problematic because most of these compounds are relatively unstable and may differ in their chemical properties. Here we describe a novel ultra-performance liquid chromatography-selected reaction monitoring mass spectroscopy (UPLC-SRM/MS) method for simultaneous quantification of key urinary eicosanoids, including the prostaglandins (PG) tetranor PGE-M, 8-iso-, and 2,3-dinor-8-iso-PGF(2α); the thromboxanes (TXs) 11-dehydro- and 2,3-dinor-TXB₂; leukotriene E₄; and 12-hydroxyeicosatetraenoic acid. In contrast to previous methods, which used time-consuming and complex solid phase extraction, we prepared samples with a simple liquid/liquid extraction procedure. Because collision-induced dissociation produced characteristic product ions for all analytes, no derivatization step for SRM/MS analysis was necessary. Analytes were separated with a short UPLC reversed-phase column (1.7 µm particles), allowing shorter run times than conventional HPLC columns. The method was validated and applied to human urine samples showing excellent precision, accuracy, detection limits, and robustness. In summary, the developed method allows robust and sensitive profiling of urinary eicosanoid species, making it a useful and valuable tool for biomarker profiling in clinical/toxicological studies.
Highlights
Eicosanoids are key mediators and regulators of inflammation and oxidative stress often used as biomarkers for diseases and pathological conditions such as cardiovascular and pulmonary diseases and cancer
Levels are elevated in Abbreviations: COX, cyclooxygenase; CVD, cardiovascular disease; HETE, hydroxyeicosatetraenoic acid; IS, internal standard; limits of detection (LOD), limit of detection; limits of quantification (LOQ), limit of quantification; LT, leukotriene; LLE, liquid-liquid extraction; PG, prostaglandin; RP, reversed-phase; SPE, solid phase extraction; SRM, selected reaction monitoring; TX, thromboxane; UPLC, ultraperformance liquid chromatography
LTE4 could be ionized in the positive mode, the negative ion mode was more advantageous for all other analytes
Summary
Eicosanoids are key mediators and regulators of inflammation and oxidative stress often used as biomarkers for diseases and pathological conditions such as cardiovascular and pulmonary diseases and cancer. Key urinary eicosanoid species produced from arachidonic acid related to proinflammatory states or oxidative stress are the following: A) tetranor PGE-M, a stable metabolite of PGE2 (present in plasma, relatively instable) synthesized by cyclooxygenase (COX)-1 and COX-2 [9, 10]. B) 8-iso-PGF2␣ ( named 8-epi-PGF2␣, 8-isoprostane, or iPF2␣-III) is probably the most popular eicosanoid molecule used as biomarker for oxidative stress because it has been validated [12] It is generated nonenzymatically by free radical-mediated oxidation of arachidonic acid [10]. Levels are elevated in Abbreviations: COX, cyclooxygenase; CVD, cardiovascular disease; HETE, hydroxyeicosatetraenoic acid; IS, internal standard; LOD, limit of detection; LOQ, limit of quantification; LT, leukotriene; LLE, liquid-liquid extraction; PG, prostaglandin; RP, reversed-phase; SPE, solid phase extraction; SRM, selected reaction monitoring; TX, thromboxane; UPLC, ultraperformance liquid chromatography. Similar to 11-dehydro-TXB2, it is assumed that increased 12-HETE levels are associated with platelet activation [26]
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