Lyso-phosphatidylethanol detected by LC-MS/MS as a potential new marker for alcohol consumption

Alcohol biomarkers are able to reflect the degree of recent or long-term alcohol consumption, covering different windows of detection. Phosphatidylethanols (PEths) are an emerging group of direct alcohol biomarkers that are widely applied in clinical and forensic applications. Their quantification can provide insight into an individual's drinking behaviour. Here, we present a new sub-class of yet unknown PEth species, LysoPEths, which are structurally related to PEth, but miss one fatty acyl chain. LysoPEths can be either a degradation product of PEth or a product of transesterification of lyso-phosphatidylcholine (LysoPC) with ethanol. To set up an analytical method, LysoPEth 16:0 was synthesised from PC 16:0/18:1 and characterised by LC-MS/MS, using an enzymatic method: phospholipase D (PLD), followed by phospholipase A₂ (PLA₂). Then, an LC-MS/MS method in MRM mode for LysoPEth 16:0 with additional LysoPEth species (LysoPEth 18:1, LysoPEth 18:2, and LysoPEth 20:4) and PEth 16:0/20:4 was developed. By incubation of freshly sampled venous blood of a teetotaller with ethanol at different concentrations, the formation of LysoPEth in parallel to PEth was investigated. With increasing ethanol concentrations, LysoPEth 16:0 was formed besides the known PEth species (PEth 16:0/18:1, PEth 16:0/18:2) for up to 72 h with LysoPEth concentrations being about three times lower than PEth concentrations. Storage of ethanol-free PEth-positive blood of an alcohol consumer at 37 °C showed that LysoPEth 16:0 concentrations increased, while PEth 16:0/18:1 concentrations decreased in the first 24 h for frozen/thawed blood, however not for freshly collected blood. Furthermore, LysoPEth 16:0 was detected in venous as well as lyophilised blood from clinical and forensic case work alongside with PEth 16:0/18:1, 16:0/18:2, and other PEth and LysoPEth species (PEth 16:0/20:4, LysoPEth 18:1, LysoPEth 18:2, and LysoPEth 20:4). LysoPEth 16:0 concentrations were found to be in linear correlation with PEth 16:0/18:1 (r²= 0.75).