A method for the detection and quantification of hydroxyl and epoxy arachidonic acid (AA) metabolites in human plasma was developed using liquid-liquid extraction, phospholipid saponification followed by derivatization of the acid moiety and liquid chromatographic tandem mass spectrometric detection. Derivatization with a pyridinium analog allowed for detection in the positive ion mode, greatly improving sensitivity and the stability of the more labile AA metabolites. The entire method utilizes a 96-well plate format, increasing sample throughput, and was optimized to measure 5-, 8-, 9-, 11-, 12-, 15-, 19-, and 20- hydroxyeicosatetraenoic acid (HETE), 5,6-, 8,9-, 11,12-, and 14,15- dihydroxyeicosatrienoic acid (DHET), and the regio- and cis-/ trans- isomers of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET). The method was validated for its applicability over the FA concentration range found in human plasma. Using 100 μL aliquots of pooled human plasma, EET levels, particularly 5,6-EET, were observed to be higher than previously reported, with measured concentrations of 23.6 ng/ml for 5,6-EET, 5.6 ng/mL for 5,6-trans-EET, 8.0 ng/mL for 8,9-EET, 1.9 ng/mL for 8,9-trans-EET, 8.8 ng/mL for 11,12-EET, 3.4 ng/mL for 11,12-trans-EET, 10.7 ng/mL for 14,15-EET, and 1.7 ng/mL 14,15-trans- EET. This method is suitable for large population studies to elucidate the complex interactions between the eicosanoids and various disease states and may be used for quantitation of a wide variety of fattyacids beyond eicosanoids from small volumes of human plasma.

建立了一种检测和定量人血浆中羟基和环氧花生四烯酸（AA）代谢物的方法，采用液液萃取、磷脂皂化、酸部分衍生化和液相色谱串联质谱检测。使用吡啶类似物进行衍生化，可以在正离子模式下进行检测，从而大大提高了更不稳定的 AA 代谢物的灵敏度和稳定性。整个方法采用 96 孔板形式，提高了样品通量，并经过优化可测量 5-、8-、9-、11-、12-、15-、19- 和 20- 羟基二十碳四烯酸 (HETE)， 5,6-、8,9-、11,12-和14,15-二羟基二十碳三烯酸(DHET)，以及5,6-、8,9-、11的区域异构体和顺式/反式异构体， 12-和14,15-环氧二十碳三烯酸（EET）。该方法在人血浆中 FA 浓度范围内的适用性经过验证。使用 100 μL 等份的混合人血浆，观察到 EET 水平（尤其是 5,6-EET）高于之前报道的水平，测得的 5,6-EET 浓度为 23.6 ng/ml，5,6-EET 浓度为 5.6 ng/mL， 6-反式-EET、8,9-EET 为 8.0 ng/mL、8,9-反式-EET 为 1.9 ng/mL、11,12-EET 为 8.8 ng/mL、11,12- 为 3.4 ng/mL反式EET、14,15-EET 为 10.7 ng/mL，14,15-反式EET 为 1.7 ng/mL。该方法适用于大规模人群研究，以阐明类二十烷酸与各种疾病状态之间的复杂相互作用，并且可用于从小体积人血浆中定量除类二十烷酸之外的多种脂肪酸。