Method development for routine liquid chromatography-mass spectrometry measurement of the alcohol biomarker phosphatidylethanol (PEth) in blood

Abstract

Background: Phosphatidylethanol (PEth) is a group of phospholipids formed from ethanol and phosphatidylcholine by action of phospholipase D. Measurement of PEth in whole blood samples is employed as an alcohol biomarker. This work aimed to further develop an LC-MS method for PEth to make it practical for routine laboratory use.

Methods: Blood samples were obtained from blood donors and from the clinical samples pool. A whole blood total lipid extract was separated on a C4 column, followed by ESI-MS detection of the deprotonated molecules in SIM mode or ESI-MS/MS detection of the major product ions (fatty acid fragments) by SRM.

Results: Initial results indicated that individual calibration curves are required for MS quantitation of some PEth forms, and that deuterated analogs are preferable over phosphatidylpropanol as the internal standard. PEth-16:0/18:1 was the single most sensitive molecular form as alcohol biomarker, being detected in every of 211 blood specimens containing 0.1-20 μmol/L total PEth at reporting limits in the range 0.1-1.0 μmol/L. PEth-16:0/18:1 and 16:0/18:2, accounting for about 36% and 26%, respectively, of the total amount, correlated well with total PEth (R(2)=0.922-0.940), but the correlation was better for the sum of both forms (R(2)=0.994). Based on analysis of specimens from 200 blood donors, 95% reference intervals (CLSI C28-A3) were estimated to be <0.70 μmol/L for total PEth, <0.20 μmol/L for PEth-16:0/18:1, and <0.18 μmol/L for PEth-16:0/18:2.

Conclusions: The LC-ESI-MS(/MS) method allowed for simultaneous qualitative and quantitative measurements of PEth forms in whole blood samples. Related to the routine application of blood PEth as alcohol biomarker, reference intervals were suggested for total PEth and the major molecular forms PEth-16:0/18:1 and 16:0/18:2.