Localization of double-bond positions in lipids by tandem mass spectrometry succeeding high-performance liquid chromatography with post-column derivatization.
Localization of double-bond positions in lipids by tandem mass spectrometry succeeding high-performance liquid chromatography with post-column derivatization.
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DOI：
10.1002/rcm.8262
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发表时间：
2018-09
期刊：
影响因子：
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通讯作者：
V. Jeck;A. Korf;Christian Vosse;H. Hayen
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文献类型：
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作者：
V. Jeck;A. Korf;Christian Vosse;H. Hayen
基本原理 脂质是一组生物分子，因其在多种生物任务中的重要性而闻名，这归因于其分子结构的多样性。脂质双键的确切位置正在影响其生物学特性。因此，其地位的确定非常重要。尽管如此，明确的任务仍然是一项具有挑战性的任务，需要开发新的方法。方法基于光化学Paternὸ Büchi 反应，提出了一种精确定位脂质中双键位置的新方法。因此，该反应是通过紫外光照射（254 nm）和额外的恒定丙酮流量进行柱后衍生化来进行的。由于丙酮与双键反应生成氧杂环丁烷而形成丙酮加合物。随后的断裂实验产生了诊断片段，允许定位双键位置。结果为了与HPLC-串联质谱在线联用，对反应时间、痕量氧以及溶剂的影响等参数进行了优化。通过 HPLC 对反应产物进行额外分离，可以进一步表征紫外线照射形成的化学结构。为了证明所开发方法的适用性，分析了磷脂异构体双（单油酰甘油）磷酸酯和二油酰磷脂酰甘油的混合物的双键位置，并对来自绿藻莱茵衣藻提取物的脂质进行了研究。结论 HPLC-串联质谱与在线反应的成功联用被证明可以定位单一脂质、脂质混合物和复杂生物样品中的双键。可以减少基质效应并获得进一步的结构信息，例如在结构异构体的情况下。这为精确定位脂质双键位置（甚至在复杂样品中）开辟了新途径。
RATIONALE Lipids are a group of biomolecules known for their importance in a broad variety of biological tasks, which is attributed to their diversity of molecular structures. The exact position of a lipids' double bond is affecting its biological properties. Therefore, the determination of its position is highly important. Nevertheless, the unambiguous assignment is still a challenging task and requires the development of new approaches. METHODS A novel method for pinpointing double bond positions in lipids is presented, based on the photochemical Paternὸ Büchi reaction. Therefore, the reaction was performed using a post-column derivatization by UV light irradiation (254 nm) and an additional constant acetone flow. The acetone adduct was formed due to the reaction of acetone with the double bond resulting in an oxetane. Subsequent fragmentation experiments generated diagnostic fragments allowing the localization of double bond positions. RESULTS Intending online hyphenation with HPLC-tandem mass spectrometry, the reaction was optimized regarding parameters such as reaction time and the influence of trace oxygen as well as solvents. An additional separation of the reaction products by means of HPLC enabled further characterization of the chemical structures formed by UV irradiation. To demonstrate the applicability of the developed method, a mixture of the constitutional phospholipid isomers bis (monooleoylglycero) phosphate and dioleyl-phosphatidylglycerol was analysed regarding their double bond positions, and lipids from an extract of the green alga Chlamydomonas reinhardtii were investigated. CONCLUSIONS The successful hyphenation of HPLC-tandem mass spectrometry with the online reaction was demonstrated for localization of double bonds in single lipids, lipid mixtures, and complex biological samples. Matrix effects can be reduced and further structural information, for example in case of constitutional isomers, can be obtained. This opens up new avenues for pinpointing lipid double bond positions even in complex samples.