The uncontrolled activation of endogenous enzymes may introduce both qualitative and quantitative artefacts when lipids are extracted from vegetal matrices. In the present study, a method based on hydrophilic interaction liquid chromatography coupled either to high-resolution/accuracy Fourier-transform mass spectrometry (HILIC-ESI-FTMS) or to linear ion trap multiple stage mass spectrometry (HILIC-ESI-MSⁿ, with n = 2 and 3) with electrospray ionization was developed to unveil one of those artefacts. Specifically, the artificial generation of methyl esters of phosphatidic acids (MPA), catalysed by endogenous phospholipase D (PLD) during lipid extraction from five oleaginous microgreen crops (chia, soy, flax, sunflower and rapeseed), was studied. Phosphatidylcholines (PC) and phosphatidylglycerols (PG) were found to be the most relevant precursors of MPA among glycerophospholipids (GPLs), being involved in a transphosphatidylation process catalysed by PLD and having methanol as a coreactant. The combination of MS² and MS³ measurements enabled the unambiguous recognition of MPA from their fragmentation pathways, leading to distinguish them from isobaric PA including a further CH₂ group on their side chains. PLD was also found to catalyse the hydrolysis of PC and PG to phosphatidic acids (PAs). The described transformations were confirmed by the remarkable decrease of MPA abundance observed when isopropanol, known to inhibit PLD, was tentatively adopted instead of water during the homogenization of microgreens. The unequivocal identification of MPA might be exploited to assess if GPL alterations are actually triggered by endogenous PLD during lipid extractions from specific vegetal tissues.

中文翻译：

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HILIC-ESI-MS 分析微绿作物脂质提取过程中人工产生的磷脂酸甲酯

当从植物基质中提取脂质时，内源酶的不受控制的激活可能会引入定性和定量的假象。在本研究中，一种基于亲水相互作用液相色谱的方法与高分辨率/准确度傅里叶变换质谱 (HILIC-ESI-FTMS) 或线性离子阱多级质谱 (HILIC-ESI-MS ⁿ ,与n = 2 和 3) 与电喷雾电离被开发来揭示这些人工制品之一。具体而言，研究了在从五种含油微绿作物（奇亚籽、大豆、亚麻、向日葵和油菜籽）中提取脂质过程中由内源性磷脂酶 D (PLD) 催化的磷脂酸甲酯 (MPA) 的人工生成。发现磷脂酰胆碱 (PC) 和磷脂酰甘油 (PG) 是甘油磷脂 (GPL) 中最相关的 MPA 前体，它们参与 PLD 催化的转磷脂酰化过程，并以甲醇作为共反应物。MS ²和 MS ³测量的组合使 MPA 能够从其碎片途径中明确识别，从而将它们与等压 PA 区分开来，包括进一步的 CH ₂组在他们的侧链上。还发现 PLD 催化 PC 和 PG 水解为磷脂酸 (PAs)。所描述的转化通过观察到的 MPA 丰度的显着降低得到证实，当在微绿色植物的均质化过程中暂时采用异丙醇代替水时，观察到的 MPA 丰度显着降低。MPA 的明确鉴定可用于评估在从特定植物组织提取脂质期间 GPL 改变是否实际上是由内源性 PLD 触发的。