Center for Translational Biomedical Research, University of North Carolina at Greensboro, North Carolina Research Campus, Kannapolis, NC, 28081, USA.
Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC, 27412, USA.
Anal Bioanal Chem. 2019 Jan;411(2):367-385. doi: 10.1007/s00216-018-1446-3. Epub 2018 Nov 8.
Oxylipins, a subclass of lipid mediators, are metabolites of various polyunsaturated fatty acids with crucial functions in regulation of systemic inflammation. Elucidation of their roles in pathological conditions requires accurate quantification of their levels in biological samples. We refined an ultra-performance liquid chromatography-multiple reaction monitoring-mass spectrometry (UPLC-MRM-MS)-based workflow for comprehensive and specific quantification of 131 endogenous oxylipins in human plasma, in which we optimized LC mobile phase additives, column, and gradient conditions. We employed heatmap-assisted strategy to identify unique transitions to improve the assay selectivity and optimized solid phase extraction procedures to achieve better analyte recovery. The method was validated according to FDA guidelines. Overall, 94.4% and 95.7% of analytes at tested concentrations were within acceptable accuracy (80-120%) and precision (CV < 15%), respectively. Good linearity for most analytes was obtained with R > 0.99. The method was also validated using a standard reference material-SRM 1950 frozen human plasma to demonstrate inter-lab compatibility. Graphical abstract ᅟ.
氧化脂类，脂类介质的一个子类，是各种多不饱和脂肪酸的代谢产物，在调节全身炎症中具有关键作用。阐明它们在病理条件下的作用需要准确量化它们在生物样本中的水平。我们改进了一种基于超高效液相色谱-多重反应监测-质谱（UPLC-MRM-MS）的工作流程，用于全面和特异性地定量人血浆中的 131 种内源性氧化脂类，其中我们优化了 LC 流动相添加剂、色谱柱和梯度条件。我们采用热图辅助策略来识别独特的转变，以提高分析物的选择性，并优化固相萃取程序以实现更好的分析物回收率。该方法根据 FDA 指南进行了验证。总体而言，在测试浓度下，94.4%和 95.7%的分析物的准确度（80-120%）和精密度（CV<15%）均在可接受范围内。大多数分析物的线性良好，R>0.99。该方法还使用标准参考物质-SRM 1950 冷冻人血浆进行了验证，以证明实验室间的兼容性。
