HILIC-ESI-MS analysis of phosphatidic acid methyl esters artificially generated during lipid extraction from microgreen crops

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.