Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15216, United States.
Department of Psychiatry, Northeast Ohio Medical University, Rootstown, OH 44272, United States.
Prostaglandins Leukot Essent Fatty Acids. 2018 Sep;136:153-159. doi: 10.1016/j.plefa.2017.01.003. Epub 2017 Jan 18.
A rapid and sensitive method using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to simultaneously quantify hydroxyeicosatetraenoic (HETE), dihydroxyeicosatrienoic (DiHETrE), epoxyeicosatrienoic acid (EET), and prostaglandin metabolites of arachidonic acid in human plasma. Sample preparation consisted of solid phase extraction with Oasis HLB (30mg) cartridges for all metabolites. Separation of HETEs, EETs, and DiHETrEs was achieved on an Acquity UPLC BEH C18, 1.7µm (100×2.1mm) reversed-phase column (Waters Corp, Millford, MA) with negative electrospray ionization mass spectrometric detection. A second injection of the same extracted sample allowed for separation and assessment of prostaglandin metabolites under optimized UPLC-MS/MS conditions. Additionally, the endogenous levels of these metabolites in five different matrices were determined in order to select the optimal matrix for assay development. Human serum albumin was shown to have the least amount of endogenous metabolites, a recovery efficiency of 79-100% and a matrix effect of 71 - 100%. Linear calibration curves ranging from 0.416 to 66.67ng/ml were validated. Inter-assay and intra-assay variance was less than 15% at most concentrations. This method was successfully applied to quantify metabolite levels in plasma samples of healthy control subjects receiving niacin administration to evaluate the association between niacin administration and eicosanoid plasma level response.
建立了一种超高效液相色谱-串联质谱法（UPLC-MS/MS），可快速灵敏地同时定量检测人血浆中羟二十碳四烯酸（HETE）、二羟二十碳三烯酸（DiHETrE）、环氧二十碳三烯酸（EET）和花生四烯酸的前列腺素代谢物。所有代谢物的样品制备均采用 Oasis HLB（30mg）小柱固相萃取法。采用 Acquity UPLC BEH C18，1.7µm（100×2.1mm）反相柱（Waters Corp，Milford，MA），在负电喷雾电离质谱检测下，实现 HETEs、EETs 和 DiHETrEs 的分离。对同一提取样品进行第二次进样，可在优化的 UPLC-MS/MS 条件下分离和评估前列腺素代谢物。此外，还测定了这五种不同基质中这些代谢物的内源性水平，以选择用于测定开发的最佳基质。人血清白蛋白显示出最少的内源性代谢物，回收率为 79-100%，基质效应为 71-100%。验证了线性校准曲线范围为 0.416-66.67ng/ml。在大多数浓度下，批内和批间变异均小于 15%。该方法成功应用于定量分析接受烟酸给药的健康对照受试者血浆样品中的代谢物水平，以评估烟酸给药与花生四烯酸血浆水平反应之间的关系。
