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Phosphatidylethanols (PEth) are formed from phosphatidylcholines and ethanol and are used as a specific and sensitive alcohol biomarker. An analytical method for analysis of PEth in oral fluid based on high-performance liquid chromatography coupled to a quadrupole tandem mass spectrometer (LC-MS/MS) was developed and validated and applied on samples collected from patients undergoing alcohol detoxification.A 200-μL aliquot of oral fluid, collected using the QuantisalTM device, was extracted with chloroform/methanol containing internal standard and subjected to LC-MS/MS analysis of three selected PEth forms (16:0/16:0, 16:0/18:1, and 16:0/18:2). Chromatographic separation was achieved on a UPLC BEH phenyl column, using a mobile phase consisting of acetonitrile and water containing 10 mmol/L ammonium hydrogen carbonate with 0.1% ammonia. The MS instrument was operated in negative electrospray ionization and selected reaction monitoring mode.The detection limit for PEth 16:0/16:0, 16:0/18:1, and 16:0/18:2 was ˜0.1 ng/mL, and the extraction recoveries at 2.0 ng/mL were in the range of 99%-114%. Method linearity over a concentration range up to 200 ng/mL was ≥0.99. No significant deviation in results was observed in an analyte stability study of two different concentrations at two different temperatures over 3 months. In 35 oral fluid samples collected from patients undergoing alcohol detoxification, the highest concentration was observed for PEth 16:0/18:1 (Detected range, 0.51-55.3 ng/mL; mean, 8.5; median, 3.1). In addition, all three PEth forms were variably identified in a majority (63%) of the oral fluid samples. The PEth 16:0/18:1 values in oral fluid showed a weak positive correlation with the corresponding values in whole blood samples (r=0.50, p=0.026, n=20).The LC-MS/MS method could reliably detect and quantify PEth in oral fluid samples collected after alcohol exposure. The method was characterized by validation data with satisfactory recovery, sensitivity, accuracy, and imprecision, and applied for analysis of clinical samples. The results suggest that measurement of PEth in oral fluid can be used as a biomarker for alcohol consumption, and as such a non-invasive complement to analysis in blood. However, further studies are required to evaluate the test characteristics (e.g. sensitivity and half-life) in comparison with PEth in blood.