Selective detection of phosphatidylethanol homologues in blood as biomarkers for alcohol consumption by LC-ESI-MS/MS

Abstract

A new validated method for the quantitation of the abnormal phospholipid phosphatidylethanol (PEth)--a biomarker for ethanol uptake--has been developed by LC-ESI-MS/MS following miniaturised organic solvent extraction and reversed phase chromatography with phosphatidylbutanol (PBut) as internal standard. PEth homologues with two fatty acid substituents-PEth 18:1/18:1, PEth 16:0/16:0-were determined in post-mortem blood collected from heavy drinkers at autopsy and also in whole blood samples from a volunteer after a single 60 g-dose of ethanol. Furthermore, PEth 18:1/16:0 or its isobaric isomer PEth-16:0/18:1 was detected. In comparison to previous high-performance liquid chromatography (HPLC) methods with evaporative light scattering detection (ELSD), the LC-MS/MS-method is more sensitive--with a limit of detection below 20 ng/ml--and more selective for single PEth homologues, while ELSD has been used for detection of the sum of PEth homologues with approximately 10 times less sensitivity. LC-MS/MS enables monitoring of PEth homologues as biomarkers for harmful and prolonged alcohol consumption as with HPLC/ELSD earlier, where PEth is measurable in blood only after more than 50 g ethanol daily intake for more than 2 weeks. Because of its higher sensitivity, there is a potential to detect single heavy drinking by LC-MS/MS, when PEth is formed in very low concentrations. This opens a new field of application of PEth to uncover single or multiple heavy drinking at a lower frequency and with a larger window of detection in blood than before by HPLC/ELSD or by use of other direct markers, e.g. ethyl glucuronide or ethyl sulfate.