Skip to main content

Identification of 48 homologues of phosphatidylethanol in blood by LC-ESI-MS/MS


Phosphatidylethanol (PEth) is an abnormal phospholipid carrying two fatty acid chains. It is only formed in the presence of ethanol via the action of phospholipase D (PLD). Its use as a biomarker for alcohol consumption is currently under investigation. Previous methods for the analysis of PEth included high-performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD), which is unspecific for the different homologues—improved methods are now based on time of flight mass spectrometry (TOF-MS) and tandem mass spectrometry (MS/MS). The intention of this work was to identify as many homologues of PEth as possible. A screening procedure using multiple-reaction monitoring (MRM) for the identified homologues has subsequently been established. For our investigations, autopsy blood samples collected from heavy drinkers were used. Phosphatidylpropanol 16:0/18:1 (internal standard) was added to the blood samples prior to liquid–liquid extraction using borate buffer (pH 9), 2-propanol and n-hexane. After evaporation, the samples were redissolved in the mobile phase and injected into the LC-MS/MS system. Compounds were separated on a Luna Phenyl Hexyl column (50 mm × 2 mm, 3 µm) by gradient elution, using 2 mM ammonium acetate and methanol/acetone (95/5; v/v). A total of 48 homologues of PEth could be identified by using precursor ion and enhanced product ion scans (EPI).

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Halter CC, Dresen S, Auwaerter V, Wurst FM, Weinmann W (2008) Kinetics in serum and urinary excretion of ethyl sulfate and ethyl glucuronide after medium dose ethanol intake. Int J Legal Med 122:123–128

    Article  Google Scholar 

  2. Dresen S, Weinmann W, Wurst FM (2004) Forensic confirmatory analysis of ethyl sulfate–a new marker for alcohol consumption–by liquid-chromatography/electrospray ionization/tandem mass spectrometry. J Am Soc Mass Spectrom 15:1644–1648

    Article  CAS  Google Scholar 

  3. Aradottir S, Asanovska G, Gjerss S, Hansson P, Alling C (2006) Phosphatidylethanol (PEth) concentrations in blood are correlated to reported alcohol intake in alcohol-dependent patients. Alcohol Alcohol 41:431–437

    CAS  Google Scholar 

  4. Varga A, Hansson P, Johnson G, Alling C (2000) Normalization rate and cellular localization of phosphatidylethanol in whole blood from chronic alcoholics. Clin Chim Acta 299:141–150

    Article  CAS  Google Scholar 

  5. Kobayashi M, Kanfer JN (1987) Phosphatidylethanol formation via transphosphatidylation by rat brain synaptosomal phospholipase D. J Neurochem 48:1597–1603

    Article  CAS  Google Scholar 

  6. Gustavsson L, Alling C (1987) Formation of phosphatidylethanol in rat brain by phospholipase D. Biochem Biophys Res Commun 142:958–963

    Article  CAS  Google Scholar 

  7. Seidler L, Kaszkin M, Kinzel V (1996) Primary alcohols and phosphatidylcholine metabolism in rat brain synaptosomal membranes via phospholipase D. Pharmacol Toxicol 78:249–253

    Article  CAS  Google Scholar 

  8. Chalifa-Caspi V, Eli Y, Liscovitch M (1998) Kinetic analysis in mixed micelles of partially purified rat brain phospholipase D activity and its activation by phosphatidylinositol 4, 5-bisphosphate. Neurochem Res 23:589–599

    Article  CAS  Google Scholar 

  9. Gunnarsson T, Karlsson A, Hansson P, Johnson G, Alling C, Odham G (1998) Determination of phosphatidylethanol in blood from alcoholic males using high-performance liquid chromatography and evaporative light scattering or electrospray mass spectrometric detection. J Chromatogr B Biomed Sci Appl 705:243–249

    Article  CAS  Google Scholar 

  10. Aradottir S, Seidl S, Wurst FM, Jonsson BA, Alling C (2004) Phosphatidylethanol in human organs and blood: a study on autopsy material and influences by storage conditions. Alcohol Clin Exp Res 28:1718–1723

    Article  CAS  Google Scholar 

  11. Welti R, Li W, Li M, Sang Y, Biesiada H, Zhou HE, Rajashekar CB, Williams TD, Wang X (2002) Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis. J Biol Chem 277:31994–32002

    Article  CAS  Google Scholar 

  12. Ruelland E, Cantrel C, Gawer M, Kader JC, Zachowski A (2002) Activation of phospholipases C and D is an early response to a cold exposure in Arabidopsis suspension cells. Plant Physiol 130:999–1007

    Article  CAS  Google Scholar 

  13. Aradottir S, Olsson BL (2005) Methodological modifications on quantification of phosphatidylethanol in blood from humans abusing alcohol, using high-performance liquid chromatography and evaporative light scattering detection. BMC Biochem 6:18

    Article  Google Scholar 

  14. Varga A, Nilsson S (2008) Nonaqueous capillary electrophoresis for analysis of the ethanol consumption biomarker phosphatidylethanol. Electrophoresis 29:1667–1671

    Article  CAS  Google Scholar 

  15. Helander A, Zheng Y (2009) Molecular species of the alcohol biomarker phosphatidylethanol in human blood measured by LC-MS. Clin Chem 55:1395–1405

    Article  CAS  Google Scholar 

  16. Gnann H, Weinmann W, Engelmann C, Wurst FM, Skopp G, Winkler M, Thierauf A, Auwarter V, Dresen S, Bouzas NF (2009) Selective detection of phosphatidylethanol homologues in blood as biomarkers for alcohol consumption by LC-ESI-MS/MS. J Mass Spectrom 44:1293–1299

    Article  CAS  Google Scholar 

  17. Ekroos K, Ejsing CS, Bahr U, Karas M, Simons K, Shevchenko A (2003) Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation. J Lipid Res 44:2181–2192

    Article  CAS  Google Scholar 

Download references


N. Ferreirós thanks the government of Basque Country for her postdoctoral grant.

Author information

Authors and Affiliations


Corresponding author

Correspondence to W. Weinmann.

Electronic supplementary material

Below is the link to the electronic supplementary material.


(PDF 875 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gnann, H., Engelmann, C., Skopp, G. et al. Identification of 48 homologues of phosphatidylethanol in blood by LC-ESI-MS/MS. Anal Bioanal Chem 396, 2415–2423 (2010).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Alcohol
  • Phosphatidylethanol
  • Phospholipids
  • Homologues
  • LC-MS/MS
  • Biomarker