Preanalytical Investigation of Polyunsaturated Fatty Acids and Eicosanoids in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry
Preanalytical Investigation of Polyunsaturated Fatty Acids and Eicosanoids in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry
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DOI：
10.1089/bio.2015.0005
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发表时间：
2016-04-01
影响因子：
1.6
通讯作者：
Ceglarek, Uta
中科院分区：
文献类型：
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作者：
Dorow, Juliane;Becker, Susen;Ceglarek, Uta
背景：分析前变量对样本矩阵有很大影响，是实验室误差的来源。近年来，低温库的作用越来越受到重视，需要系统的研究。花生四烯酸的代谢是一种有用的质量标志，因为二十烷类化合物在体外容易受到氧化过程的影响。材料和方法：采用在线固相萃取-液-质联用法分析多不饱和脂肪酸(PUFA)及其相关代谢物。研究了不同的血浆抗凝剂以及血清、冻融循环(n=5)、4℃短期保存、室温长达120分钟以及-20℃、-80℃和-150℃长期保存至180天的影响。结果：多不饱和脂肪酸代谢产物在乙二胺四乙酸(EDTA)稳定的全血中稳定120min，在EDTA稳定的血浆中稳定30min。在蛋白质耗尽后，血浆在4摄氏度下的稳定性可以进一步提高到7天，而添加抗氧化剂，如丁基羟基甲苯或氮气覆盖没有保护作用。反复冻融循环(n>；1)导致二十烷类化合物的形成高达63%。在零下20摄氏度的长期储存导致30天后二十烷类化合物的大幅增加，这可以通过在储存前消耗蛋白质来防止。在-80摄氏度和-150摄氏度的低温储存显示，180天后八种二十烷类化合物的浓度降低。与常规冷冻法相比，液氮冲击冷冻法的优势还不能得到证实。结论：为了将体外数据的变异性降到最低，需要为人体基质中二十烷类化合物的分析建立明确的分析前条件。
Background: Preanalytical variables have a great impact on sample matrices and are a source of laboratory errors. The effect of cryobanking, which is gaining great importance recently, requires systematic investigation. The arachidonic acid metabolism is useful as a quality marker since eicosanoids are easily subjected to in vitro oxidation processes.Materials and Methods: Polyunsaturated fatty acids (PUFAs) and related metabolites were analyzed by online solid-phase extraction coupled to liquid chromatography-tandem mass spectrometry. The influence of different plasma anticoagulants, as well as serum, freeze-thaw cycles (n = 5), short-term storage at 4 degrees C, room temperature up to 120 minutes, and long-term storage at -20 degrees C, -80 degrees C, and -150 degrees C up to 180 days, were investigated. We further investigated the influence of protein depletion, antioxidants, and shock-freezing on plasma.Results: PUFA metabolites were stable at 4 degrees C in ethylenediaminetetraacetic acid (EDTA)-stabilized whole blood for 120 minutes and in EDTA-plasma for 30 minutes. Plasma stability at 4 degrees C could be further increased up to 7 days after protein depletion, while addition of antioxidants such as butylated hydroxytoluene or coverage with nitrogen had no protective effects. Repeated freeze-thaw cycles (n > 1) resulted in eicosanoid formation up to 63%. Long-term storage at -20 degrees C led to substantial eicosanoid increases after 30 days, which could be prevented by depleting proteins before storage. Cryobanking at -80 degrees C and -150 degrees C revealed decreased concentrations of eight eicosanoids after 180 days. An advantage of shock-freezing with liquid nitrogen could not be confirmed compared to conventional freezing.Conclusion: Defined preanalytical conditions for eicosanoid analysis in human matrices are required to minimize in vitro data variability.