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

ABSTRACT 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 ( R 2 > 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 ( R 2 ≥ 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.