Molecular Species of the Alcohol Biomarker Phosphatidylethanol in Human Blood Measured by LC-MS

Abstract

Background: The alcohol biomarker phosphatidylethanol (PEth) comprises a group of ethanol-derived phospholipids formed from phosphatidylcholine by phospholipase D. The PEth molecular species have a common phosphoethanol head group onto which 2 fatty acid moieties are attached. We developed an electrospray ionization (ESI) LC-MS method for qualitative and quantitative measurement of different PEth species in human blood.Methods: We subjected a total lipid extract of whole blood to HPLC gradient separation on a C4 column and performed LC-ESI-MS analysis using selected ion monitoring of deprotonated molecules for the PEth species and phosphatidylpropanol (internal standard). Identification of individual PEth species was based on ESI–tandem mass spectrometry (MS/MS) analysis of product ions.Results: The fatty acid moieties were the major product ions of PEth, based on comparison with PEth-16:0/16:0, 18:1/18:1, and 16:0/18:1 reference material. For LC-MS analysis of different PEth species in blood, we used a calibration curve covering 0.2–7.0 μmol/L PEth-16:0/18:1. The lower limit of quantitation of the method was <0.1 μmol/L, and intra- and interassay CVs were <9% and <11%. In blood samples collected from 38 alcohol patients, the total PEth concentration ranged between 0.1 and 21.7 μmol/L (mean 8.9). PEth-16:0/18:1 and 16:0/18:2 were the predominant molecular species, accounting for approximately 37% and 25%, respectively, of total PEth. PEth-16:0/20:4 and mixtures of 18:1/18:1 plus 18:0/18:2 (not separated using selected ion monitoring because of identical molecular masses) and 16:0/20:3 plus 18:1/18.2 made up approximately 13%, 12%, and 8%.Conclusions: This LC-MS method allows simultaneous qualitative and quantitative measurement of several PEth molecular species in whole blood samples.