Methodological modifications on quantification of phosphatidylethanol in blood from humans abusing alcohol, using high-performance liquid chromatography and evaporative light scattering detection

BACKGROUND: Phosphatidylethanol (PEth) is an abnormal phospholipid formed slowly in cell membranes by a transphosphatidylation reaction from phosphatidylcholine in the presence of ethanol and catalyzed by the enzyme phospholipase D. PEth in blood is a promising new marker of ethanol abuse depending on the high specificity and sensitivity of this marker. None of the biological markers used in clinical routine at the present time are sensitive and specific enough for the diagnosis of alcohol abuse. The method for PEth analysis includes lipid extraction of whole blood, a one-hour HPLC separation of lipids and ELSD (evaporative light scattering) detection of PEth. RESULTS: Methodological improvements are presented which comprise a simpler extraction procedure, the use of phosphatidylbutanol as internal standard and a new algorithm for evaluation of unknown samples. It is further demonstrated that equal test results are obtained with blood collected in standard test tubes with EDTA as with the previously used heparinized test tubes. The PEth content in blood samples is stable for three weeks in the refrigerator. CONCLUSION: Methodological changes make the method more suitable for routine laboratory use, lower the limit of quantification (LOQ) and improve precision.