Development and Optimization of an LC-MS/MS Method for Quantification of Phosphatidylethanol in Human Whole Blood

Phosphatidylethanols (PEth) are ethanol-derived phospholipids formed in red blood cell membranes during alcohol exposure and have emerged as highly specific biomarkers for recent alcohol use. Their extended 2-4-week detection window makes them uniquely valuable for objective monitoring in liver disease and transplantation, where accurate assessment of alcohol abstinence is critical. We developed and validated a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of PEth 16:0/18:1 and PEth 16:0/18:2 in whole blood. Multiple sample extraction strategies were evaluated to optimize recovery, sensitivity, and workflow practicality. The final method employed phospholipid-removal cartridges in a reversed-use configuration to retain, rather than remove, phospholipids. Wash and elution conditions were systematically optimized to achieve adequate sensitivity without a post-extraction evaporation step, enabling direct LC-MS/MS loading after elution and automation compatibility. Due to detectable and lot-variable endogenous PEth in commercial human blood products, multiple matrix sources were evaluated. Chicken whole blood demonstrated the cleanest background and was selected for calibrator and quality-control preparation. The developed method achieved linearity from 10 to 1000 ng/mL (R² > 0.99) with limits of quantification below 10 ng/mL, and within- and between-run precision of < 10% and < 12%, respectively. Method comparison against a national reference laboratory showed excellent agreement (R² ≥ 0.93; bias within ± 5%). No significant carryover, ion suppression, or lipid interference was observed. This developed and optimized LC-MS/MS method provides a sensitive, simplified, and automation-compatible approach for PEth 16:0/18:1 and 16:0/18:2 quantification, well-suited for high-throughput implementation in clinical laboratories.