Schmidt Louis, Burmeister Laura Sophie, Greinacher Andreas, König Stefanie, Garscha Ulrike
Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Greifswald University, 17489 Greifswald, Germany.
Institute of Transfusion Medicine, University Medicine Greifswald, 17489 Greifswald, Germany.
Metabolites. 2022 Nov 30;12(12):1198. doi: 10.3390/metabo12121198.
Eicosanoids are lipid mediators generated from arachidonic acid with pro- and anti-inflammatory properties. Despite these lipid mediators being known for decades, quantitative determination in biological samples is still challenging due to low abundance, instability, the existence of regio- and stereoisomers, and a wide polarity range that hampers chromatographic separation. In this study, we developed a supercritical fluid chromatography mass spectrometry (SFC-MS) platform for the quantification of relevant eicosanoids. Application of a chiral amylose-based column and modifier combination of 2-propanol/acetonitrile offered separation and sufficient resolution of 11 eicosanoids (5-, 12-, 15-HETE, PGB, LTB, t-LTB, 20-OH-LTB, PGE, PGD, PGF, TxB) with baseline separation of isobaric analytes within 12 min. The method was validated in terms of range (78-2500 ng/mL), linearity, accuracy, precision, and recovery according to EMA guidelines. Finally, we confirmed the method's applicability by quantifying eicosanoid levels in human primary blood cells. In conclusion, we present a validated SFC-MS method for the determination of relevant eicosanoids in biological samples with a wide range of polarity while maintaining baseline separation of isobars, which allows coupling to a single quadrupole mass detector.
类二十烷酸是由花生四烯酸产生的具有促炎和抗炎特性的脂质介质。尽管这些脂质介质已为人所知数十年，但由于其丰度低、不稳定、存在区域异构体和立体异构体以及极性范围广阻碍色谱分离，在生物样品中的定量测定仍然具有挑战性。在本研究中，我们开发了一种超临界流体色谱质谱联用（SFC-MS）平台用于相关类二十烷酸的定量分析。使用基于直链淀粉的手性柱和2-丙醇/乙腈的改性剂组合，可在12分钟内实现11种类二十烷酸（5-、12-、15-羟基二十碳四烯酸、前列腺素B、白三烯B、反式白三烯B、20-羟基白三烯B、前列腺素E、前列腺素D、前列腺素F、血栓素B）的分离和足够的分辨率，同量异位分析物实现基线分离。该方法根据欧洲药品管理局（EMA）指南在范围（78 - 2500 ng/mL）、线性、准确度、精密度和回收率方面进行了验证。最后，我们通过定量人原代血细胞中的类二十烷酸水平证实了该方法的适用性。总之，我们提出了一种经过验证的SFC-MS方法，用于测定生物样品中具有广泛极性的相关类二十烷酸，同时保持同量异位素的基线分离，这允许与单四极杆质量检测器联用。
