Quantification of Phosphatidylethanols in Whole Blood as a Proxy for Chronic Alcohol Consumption, Using Ultra Performance Convergence Chromatography Tandem Mass Spectrometry.

Background: Detection of alcohol consumption after a longer period can be useful in certain patient groups. To monitor chronic alcohol consumption, a novel analytical method for the quantification of phosphatidylethanols (PEths) was developed and validated using ultra performance convergence chromatography-tandem mass spectrometry.

Methods: The main phosphatidylethanols like palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol (POPEth), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanol, and 1,2-dioleoyl-sn-glycero-3-phosphoethanol were analyzed using a simple and fast sample preparation protocol followed by chromatographic separation using ultra performance convergence chromatography, a novel kind of supercritical fluid chromatography. Mass spectrometric detection was conducted by applying negative electrospray ionization and multiple reaction monitoring mode. Only 50 μL of whole blood is needed for the simultaneous quantification of all 3 compounds within 5-minute run-to-run analysis time. POPEth-d5 was applied as internal standard.

Results: The method was validated according to the Food and Drug Administration guidelines. Correlation coefficients were higher than 0.995 for all 3 compounds. Intraday and interday inaccuracies were <15% for all analytes in the established linear range. Intraday and interday imprecision were <15% for all analytes. Lower limit of quantification for 1,2-dioleoyl-sn-glycero-3-phosphoethanol, palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanol, and POPEth are, respectively, 3, 6, and 6 mcg/L. Sample stability at -80°C was 1 year. Extracts were stable for 1 day in the autosampler and 2 days at 2-8°C in a closed Eppendorf tube. Samples were tested after 3 freeze-thaw cycles and considered stable. Patient samples have been analyzed with this new method. In a cohort of 248 pregnant women, 17 patients (6.9%) scored positive for PEth.

Conclusions: The described method is suitable for the simultaneous quantitative analysis of the most abundant PEth homologues. Major advantages are low LLOQs, minimal sample volume and clean-up, and a short run time. The method is now available to monitor alcohol consumption in patients and has been incorporated in clinical practice and research.