Molecular species of the alcohol biomarker phosphatidylethanol in human blood measured by LC-MS.
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 micromol/L PEth-16:0/18:1. The lower limit of quantitation of the method was <0.1 micromol/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 micromol/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.
Helander
Anders
A
Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden. anders.helander@ki.se
Zheng
Yufang
Y
D000428:Alcohol Drinking / Q000097:blood*; D015415:Biomarkers / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D020404:Glycerophospholipids / Q000097:blood*; D006801:Humans; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D015415:Biomarkers; D020404:Glycerophospholipids; C051521:phosphatidylethanol