BACKGROUND Reactive oxygen species (ROS) are produced in the body during normal metabolism by means of enzymes and non-enzymatic chemical reduction of molecular oxygen. In case of the prevalence of ROS formation over their elimination, highly reactive free radicals can be accumulated and can cause multiple damages to the biomolecules and cells. Determination of isoprostanes in biological matrices is most often used to register free radical damage and requires selective, sensitive and specific techniques. METHODS This study presents the development and validation of the LC-MS/MS method for the determination of 8-iso-Prostaglandin F2α in human plasma utilising a modified liquid-liquid extraction procedure with phase separation. RESULTS Modified sample preparation procedure assured higher extraction yield, clear separation of organic layer from the plasma water phase and protein precipitates, and better-purified product for instrumental analysis. Linearity was validated in the range 0.1-5.0 µg/L with R2 > 0.996; normalised matrix varied between 86.0% and 108.3%, accuracy ranged from 90.4 % to 113.9% and precision both within runs and between runs was less than 7%. With a run time of 10 min, a throughput of over 50 samples per working day could be performed. CONCLUSIONS The method meets all the current industrial validation criteria and allows the accurate and precise determination of 8-iso-PGF2α in human plasma at diagnostically significant concentration range.

中文翻译：

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相分离液-液萃取，通过 LC-MS/MS 定量人血浆中的 8-异前列腺素 F2 Alpha。


背景技术活性氧(ROS)是在正常代谢期间通过分子氧的酶和非酶化学还原在体内产生的。如果活性氧的形成超过其消除，高反应性自由基就会积聚，并对生物分子和细胞造成多重损害。生物基质中异前列烷的测定最常用于记录自由基损伤，需要选择性、灵敏和特定的技术。方法 本研究介绍了 LC-MS/MS 方法的开发和验证，该方法利用改进的相分离液液萃取程序测定人血浆中的 8-异前列腺素 F2α。结果 改进的样品制备程序确保了更高的提取率、有机层与血浆水相和蛋白质沉淀物的清晰分离，以及用于仪器分析的更好纯化的产品。在 0.1-5.0 µg/L 范围内验证线性，R2 > 0.996；归一化矩阵的变化范围为 86.0% 至 108.3%，准确度范围为 90.4% 至 113.9%，运行内和运行间的精度均小于 7%。运行时间为 10 分钟，每个工作日可处理超过 50 个样品。结论 该方法满足当前所有工业验证标准，并且可以在具有诊断意义的浓度范围内准确、精确地测定人血浆中的 8-iso-PGF2α。