University of Teramo, Faculty of Bioscience and Technology for Food, Agriculture and Environment, 64100 TE, Italy.
Sapienza University of Rome, Department of Chemistry, 00185 RM, Italy.
J Pharm Biomed Anal. 2020 Jul 15;186:113302. doi: 10.1016/j.jpba.2020.113302. Epub 2020 Apr 17.
Oxidative stress is a pathological condition characterized by an imbalance between body's antioxidant defenses and oxidizing agents, resulting in damage of endogenous molecules. These products can be used as markers of oxidative conditions; in particular, isoprostanes (IsoPs) come from the reaction of arachidonic acid with reactive oxygen species (ROS) and are currently defined as gold markers of oxidative stress in urine. Our main goal was the development of a reliable analytical method for the determination and quantification of the IsoPs in human urine by dispersive Liquid-Liquid Micro Extraction (dLLME) coupled with micro Solid Phase Extraction (μSPE) clean-up and HPLC-MS/MS analysis. The selected compounds are present in very small concentration in urine, furthermore, due to relevant matrix effect, they are challenging for ESI-MS/MS analysis. This approach provided selectivity and sensitivity for 8-isoprotaglandine F2α (8-iso-PGF2α), the "gold" OS marker, together with the main isomers. dLLME extraction allowed a significant enrichment factor and μSPE clean-up provided the removal of ion-suppressing compounds from the sample resulting in low matrix effect. The chromatographic separation was also challenging as the target compounds possess very similar chemical characteristics, so experimental conditions were carefully tuned. The reported method represents a useful tool for the detection of IsoPs in urine taking advantage of the combination of dLLME extraction and μSPE clean-up; overall recoveries were above 50 % and matrix effects were ≤15 %, with LOQs ranging between 0.020 and 0.060 ng mL. The procedure is easy to use and rapid allowing the removal of interfering compounds and matrix effect maintaining a highly sensitive determination.
氧化应激是一种病理状态，其特征是体内抗氧化防御和氧化剂之间的不平衡，导致内源性分子受损。这些产物可以作为氧化条件的标志物；特别是，类异戊二烯（IsoPs）来自花生四烯酸与活性氧（ROS）的反应，目前被定义为尿液中氧化应激的金标志物。我们的主要目标是开发一种可靠的分析方法，通过分散液 - 液微萃取（dLLME）与微固相萃取（μSPE）净化和 HPLC-MS/MS 分析来测定和定量人尿中的 IsoPs。所选化合物在尿液中的浓度非常低，此外，由于相关的基质效应，它们对 ESI-MS/MS 分析具有挑战性。这种方法为 8-异前列腺素 F2α（8-iso-PGF2α），即“金”OS 标志物，以及主要异构体提供了选择性和灵敏度。dLLME 萃取允许显著富集因子，μSPE 净化去除样品中的离子抑制化合物，从而降低基质效应。由于目标化合物具有非常相似的化学特性，因此色谱分离也具有挑战性，因此仔细调整了实验条件。该方法代表了一种利用 dLLME 萃取和 μSPE 净化相结合检测尿液中 IsoPs 的有用工具；总体回收率高于 50％，基质效应≤15％，LOQs 在 0.020 和 0.060ng/mL 之间。该程序易于使用且快速，可去除干扰化合物和基质效应，同时保持高度灵敏的测定。
