RESUMO
Phosphatidylethanol (PEth) is a group of aberrant phospholipids formed in cell membranes in the presence of ethanol by the catalytic action of the enzyme phospholipase D on phosphatidylcholine. Recently published literature has demonstrated the existence of several molecular species of PEth in samples drawn from alcohol-dependent subjects. A novel liquid chromatography high-resolution mass spectrometry (LC-HRMS) method coupled with a lipidomic strategy was developed and validated for the quantitative profiling of PEth molecular species in human blood collected from heavy and social drinkers. Chromatography was performed on a C18 column using acetonitrile, 10mM ammonium acetate, and 2-propanol as mobile phases with a 22-min gradient. HRMS experiments were performed on an LTQ-Orbitrap XL hybrid mass spectrometer equipped with an electrospray ionization source operated in negative ion mode. The theoretical masses of [M-H](-) of PEth species were calculated from the elemental chemical formula by varying the length and unsaturation grade of the fatty acid side chains; identification of PEth species in blood was achieved by searching the accurate masses of the targeted compounds in the acquired full-scan LC-HRMS chromatogram. The chemical structure of tentatively identified PEth species was elucidated through HR multiple mass experiments. The validated LC-HRMS method was selective, as warranted by HRMS at 60,000 resolution and 4 ppm accuracy. Linearity was observed in the 0.001-2.000 µM range, and limit of detection of 0.0005 µM and limit of quantitation of 0.001 µM were obtained for single PEth species. Imprecision and inaccuracy were always lower than 10% and 15%, respectively. The identification capabilities of the method were tested on blood samples collected from heavy drinkers (n=11), social drinkers (n=8), and teetotalers (n=10). The high sensitivity of the method led to the simultaneous identification of 17 different PEth molecular species in blood collected from heavy drinkers, and 2 PEth species (16:0/18:1 and 16:0/18:2) in blood collected from social drinkers.