Validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for erythrocyte phosphatidylethanol revealing critical considerations for its use as a clinical biomarker

• PEth is a red cell membrane phospholipid used for ethanol abstinence monitoring. • Different extraction solvents may cause under-reporting of results. • Classical recovery experiments may be insufficient for membrane derived lipids. • This work shows that extraction solvent choice can alter reported PEth values. • Cut-off concentrations for PEth must be reflective of extraction methodology. Erythrocyte membrane-incorporated phosphatidylethanol (PEth) forms only in the presence of ethanol and, once formed, provides a persisting marker for historical alcohol consumption. Relationships between PEth concentration, extent of consumption and time from consumption are under investigation. Threshold values of PEth have been proposed as indicators for any, or for harmful alcohol consumption. Here, we describe an assay for erythrocyte PEth 16:0/18:1 that offers the efficiency needed for routine clinical deployment, in the context of a fully validated methodology. However, we observe that conventional procedures for validating assay methodology are insufficient where the analyte of interest, membrane-incorporated PEth 16:0/18:1, has different physicochemical properties to the soluble PEth 16:0/18:1 and PEth 16:0/18:1-d5 that are used for making calibrator, controls and internal standards. Whereas the internal standard did fully correct for differences in matrix effects and recovery when different extraction solvents were applied to calibrators and controls (in soluble form), it failed to correct for a 1.5-fold difference in the relative efficiency of two solvents, in this case, acetonitrile and isopropanol in extracting PEth from erythrocyte membrane in clinical samples. Differences in the efficiency of the extraction of membrane-bound PEth translate to different results from the same specimen. That can mean that threshold values derived by one methodology cannot be safely generalised to another. That hampers the generalisability of individual laboratory’s experience with PEth assay results. Harmonising extraction methodology between laboratories becomes very important where membrane-incorporated PEth itself remains unavailable as an assay standard.