Systematic Study on Phosphatidylethanol (PEth) Stability and De Novo Formation in Authentic Whole Blood Samples

Phosphatidylethanols (PEth) are a group of abnormal phospholipids that are only formed in the presence of ethanol. Particularly, PEth 16:0/18:1 is a popular marker for ethanol abstinence monitoring in the 2-4 weeks preceding blood collection. While a number of studies previously investigated the long-term stability of PEth 16:0/18:1 in whole blood, they came to conflicting results. An analytical liquid chromatography-tandem-mass spectrometry-based method for the quantification of PEth 16:0/18:1 and 16:0/18:2 was fully validated and used to study the long-term stability (up to 5 months) at different storage conditions (room temperature [RT], 4°C, -20°C, and -80°C) as well as stability during postal transport. PEth 16:0/18:1 was found to be stable until the end of the study period when stored at -20°C and -80°C but only showed acceptable stability for 1 month at 4°C and 7 days at RT. In accordance with previous studies, PEth 16:0/18:2 degraded rapidly during transport and storage at RT. For the first time, PEth de novo formation was investigated using whole blood samples from a controlled drinking experiment with and without the in vivo presence of ethanol (instead of spiked samples or animal experiments). Significant PEth 16:0/18:1 de novo formation when stored at -20°C and RT as early as 14 h after sample collection was found, which crucially highlights the importance of proper sample storage. Storage at -20°C should be prevented due to the potential for rapid de novo formation. Ideally, storage (short- and long-term) at -80°C is preferred for reliable results.