Development and application of an LC-MS method for the alcohol biomarker phosphatidylethanol (PEth) in blood

Abstract: Objective biomarkers tracing alcohol consumption are demanded in many situations when alcohol drinking is in focus, e.g. during monitoring of patient in a treatment program, in forensic medicine, workplace testing or biochemical validation of selfreport in research. Phosphatidylethanol (PEth) is an abnormal phospholipid formed only in the presence of ethanol that can be used as a sensitive and specific alcohol biomarker to detect current risky alcohol consumption. The aim of this project was to develop an liquid chromatography-mass spectrometry (LC-MS) method for PEth that is suitable for routine use. PEth was extracted from whole blood and separated by LC-MS using a C4 column in a reversed phase system by gradient elution. The limit of detection (S/N ≥ 3) and limit of quantification (S/N ≥ 10) were ≤ 0.02 and ≤ 0.1 μmol/L, respectively. The calibration curve was linear in the concentration range 0.2-20 μmol/L and the intraassay CV % for total PEth was ≤ 8.6 % and the inter- assay CV was < 11 %. The CV was lower using isotope labeled PEth as internal standard in the MS/MS mode. Nine of the most common PEth forms were evaluated by both LC-MS and LCMS/ MS. PEth-16:0/18:1 and PEth-16:0/18:2 were found to be the major forms in blood from alcoholic patients. The correlations of PEth-16:0/18:1 and PEth-16:0/18:2 to total PEth were good (R2 = 0.973) and PEth-16:0/18:2 (R2= 0.983) but together they correlated even better with total PEth. In 200 blood samples from blood donors and 3023 from the routine pool, the majority had a total PEth concentration ≤ 0.5 μmol/L. The amount of PEth formed in whole blood samples that were incubated in the presence of ethanol varied considerably between individuals. The value of PEth as an alcohol biomarker was compared with ethyl glucuronide (EtG), ethyl sulfate (EtS) and carbohydrate deficient transferrin (CDT) in an outpatient treatment program for alcohol-dependent subjects. Compared with CDT, PEth was found to be a more sensitive biomarker. In conclusion, a sensitive and specific LC-MS method was developed for the routine measurement of PEth in whole blood samples. The measurement of PEth- 16:0/18:1 alone or in combination with PEth-16:0/18:2 did not affect test sensitivity compared with total PEth. The use of PEth in combination with other biomarker is preferred, due to inter-individual variation in PEth formation.