Determination by UHPLC‐QTOF‐HRMS of Phosphatidylethanol (PEth) in Dried Blood Spots: Method Validation and Practical Application of a Rising Alcohol Abuse Biomarker With Minimally Invasive Sampling

The goal of our study was to develop and validate a simple, quick, and sensitive method to detect phosphatidylethanol (PEth) in dried blood spots (DBS). A 30-μL aliquot of blood was collected on a DBS card and allowed to dry at room temperature. Then, the spot was cut and transferred into a clean tube where the internal standard (PEth-D5) and 1-mL hexane were added followed by stirring, sonication, and centrifugation at room temperature. The dried supernatant was reconstituted with 30-μL acetonitrile and analyzed by UHPLC-HRMS-QTOF. Calibration curve was created at 20, 50, 100, 200, 300, and 500 ng/mL; the limit of detection was calculated at 5 ng/mL (S/N > 3) while accuracy, precision, recovery, and matrix effect were successfully evaluated, along with the analyte stability at different time intervals and temperatures. The study demonstrates that quantifying PEth 16:0/18:1 from DBS cards is feasible using UHPLC-QTOF or UHPLC-QqQ instrumentation while the QTOF method was validated and proved reliable for PEth detection to assess both excessive alcohol consumption and alcohol abstinence, matching current guidelines. Preliminary data on authentic samples confirmed the method's performance in terms of ease, sustainability, and speed, supporting its great potential for routine toxicological diagnosis of chronic alcohol abuse.