Phosphatidylethanol (Peth), a group of aberrant phospholipids formed in cell membranes in the presence of ethanol, has been recently proposed as biomarker of chronic alcohol abuse. The aim of this study was to develop a new analytical method, based on NACE online coupled with a mass spectrometer for the analysis of Peth in blood For this purpose an ion-trap mass spectrometer equipped with an orthogonal ESI source operating in negative ion mode was used An alkaline solution of ammonium acetate 5 mM (pH 9) in water/methanol (MeOH) (80 20 v/v) was delivered as coaxial sheath liquid All experiments were performed using an uncoated fused-silica capillary (90 cm x 75 mu m id) The effects of variable percentages of ACN, MeOH, 2-propanol, dichloromethane, along with variable concentrations of ammonium acetate were investigated for the separation of Peth Collectively, a separation medium composed of ACN (45% v/v), 2-propanol (20% v/v), dichloromethane (20% v/v), MeOH (10% v/v), water (5% v/v), and ammonium acetate (25 mM) was chosen The estimated LOD was 0 1 mu M, while LOQ was 0 4 mu M. Within-run (ultra-day) and between-run (inter-day) precision was always lower than 15% The method proved to be robust and reliable. The MS detector allowed the simultaneous identification of several Peth homologues, and the use of an internal standard (phosphatidylbutanol) with similar electrophoretic properties of that of Peth increased quantitation effectiveness