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Clinical Chemistry 0: clinchem.2008.120923v1, 2009; 10.1373/clinchem.2008.120923
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Received on November 19, 2008
Accepted on April 23, 2009

Lipids, Lipoproteins, and Cardiovascular Risk Factors

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

Anders Helander 1* Yufang Zheng 1

1 Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden

* To whom correspondence should be addressed. E-mail: anders.helander{at}ki.se.

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.




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Copyright © 2009 by the American Association for Clinical Chemistry.