A new sample preparation approach for routine liquid chromatography–tandem mass spectrometry measurement of the alcohol biomarker phosphatidylethanol and its main isoforms in blood

Phosphatidylethanols (PEths) are currently under investigation as highly sensitive and specific direct biomarkers of long term alcohol abuse. PEths belong to a group of aberrant phospholipids formed in erythrocyte membranes in presence of ethanol by the catalytic action of the enzyme phospholipase D on phosphatidylcholine. To monitor alcohol consumption, an analytical method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) with novel and automated sample preparation was developed and validated for the simultaneous quantification of PEth 16:0/18:1, PEth 16:0/16:0 and PEth 18:1/18:1 in whole blood samples. Prior to detection, an important practical aspect in the work-flow of PEths analysis is the sample preparation step. To date, traditional techniques such as liquid-liquid extraction (LLE) and solid phase extraction (SPE) requires multiple steps to remove blood interferences. Due to the simplicity of use and the possibility of automation, samples filtration is also a widespread technique in biomedical laboratories. In this work, a reliable sample preparation method uses Phree™ Phospholipid Removal Plates (Phenomenex, California, USA) based on an automated filtration was developed to extract PEths from human whole blood. Surface characteristics of Phospholipids Removal material allow phospholipids retention on the filter and a suitable PEths recovery after elution. The blood samples were added with internal standard (IS) and purified in acetonitrile (1mL). After centrifugation, the supernatants were applied to the Phospholipids Removal Plates in an automated workstation. After washing, the phospholipids retained on the filter were eluted with 1-mL methanol 1% ammonia. PEth 16:0/18:1, PEth 16:0/16:0 and PEth 18:1/18:1 were extracted using the proposed method and detected by LC–MS/MS operated in electron spray ionization (ESI). The detection of all compounds was based on multiple reaction monitoring (MRM) transitions. This method was validated for the quantitative profiling of PEth molecular species in human blood collected from heavy and social drinkers. Further investigations are necessary, to establish cut-off levels for PEths as diagnostic markers for the determination of drinking habits like abstinence, social or heavy drinking.