University of Tübingen, Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Department of Cardiology and Angiology, University Hospital Tübingen, Otfried-Müller-Strasse 10, 72076, Tübingen, Germany.
J Pharm Biomed Anal. 2020 Sep 10;189:113426. doi: 10.1016/j.jpba.2020.113426. Epub 2020 Jun 20.
Oxylipins play an important role in cell signaling and they act as auto- and paracrine factors. There are numerous reports on the analysis of oxylipins in biofluids, especially in plasma. Only a limited number of studies addressed the analysis of oxylipins in platelets using modern, sensitive LCMS methods, even though these compounds have a huge impact on platelet functions and thrombo-inflammation. In this work, a new method based on superficially porous particle (2.7 μm) capillary column (0.5 mm ID) and micro-liquid chromatography coupled to tandem mass spectrometry (μUHPLC-ESI-QqQ-MS/MS) method has been developed, optimized and validated. It has finally been successfully applied for human plasma and platelet analysis. The method allows the precise and accurate simultaneous quantification of 42 oxylipins with 13 deuterated internal standards. Solid phase extraction with Bond Elut Certify II provides good extraction recoveries (on average around 75 %). The μUHPLC-MS/MS method is selective, sensitive (LOQs between 30 and 150 pg/mL) and shows good linearity. Limits of detections for most of the compounds are between 2 and 250 fmol on column. Twenty-three oxylipins have been detected in plasma and 19 in non-activated (resting) platelets (all samples were from healthy donors). The μUHPLC-MS/MS method uses very low volume of mobile phase (less than 250 μL of organic solvents in mobile phase per analysis), and therefore is considered environmentally friendly. It also turned out to be robust enough for routine analysis.
氧化脂类在细胞信号转导中发挥重要作用，它们作为自分泌和旁分泌因子。有大量关于生物流体中氧化脂类分析的报告，特别是在血浆中。尽管这些化合物对血小板功能和血栓炎症有巨大影响，但只有有限数量的研究使用现代、敏感的 LCMS 方法来分析血小板中的氧化脂类。在这项工作中，开发、优化和验证了一种基于表面多孔颗粒（2.7μm）毛细管柱（0.5mm ID）和微液相色谱-串联质谱（μUHPLC-ESI-QqQ-MS/MS）方法的新方法。最终成功应用于人血浆和血小板分析。该方法允许使用 13 个氘代内标物精确、准确地同时定量 42 种氧化脂类。使用 Bond Elut Certify II 固相萃取可提供良好的提取回收率（平均约为 75%）。μUHPLC-MS/MS 方法具有选择性、灵敏度（LOQs 在 30 至 150pg/mL 之间）和良好的线性。大多数化合物的检测限在柱上为 2 至 250fmol。在血浆中检测到 23 种氧化脂类，在非激活（静止）血小板中检测到 19 种（所有样品均来自健康供体）。μUHPLC-MS/MS 方法使用的流动相体积非常小（每次分析中流动相中的有机溶剂少于 250μL），因此被认为是环保的。它还足够稳健，可以进行常规分析。
