A new CE-ESI-MS method for the detection of stable hemoglobin acetaldehyde adducts, potential biomarkers of alcohol abuse.

A new CE-ESI-MS method was developed to provide a simple way to study changes to hemoglobin (HbA) induced by acetaldehyde (Ach) in vitro. Instrumental parameters were univariately optimized in order to maximize the sensitivity of the CE-ESI-MS method. The electrophoretic separations were carried out in poly-E323-coated capillaries using 60 mM formic acid raised to pH 3.0 with ammonia and containing 5% 2-propanol while the sheath liquid, 2-propanol/water (30:70) with 0.1% formic acid, was delivered at 1.0 microL/min through a coaxial sheath flow electrospray interface. The HbA was incubated with Ach for intervals up to 24 h at concentration varying in the window 0.2-20 mM. Four stable Ach-hemoglobin adducts in the hemoglobin tryptic digest were observed at the submillimolar Ach concentration and characterized by MS/MS experiments: although the alpha and beta N-amino terminal modifications were expected, the two internal ones arising, respectively, from the condensation of Ach molecules on the histidine residue in position 4 in alpha4 (i.e. the fourth peptide after tryptic digestion of alpha chain starting from amino terminal) and on the asparagine residue in position 2 in beta3, were identified for the first time. During the in vitro experiments higher concentrations of Ach were also used; however, it was not possible to identify any other stable modification of hemoglobin. Interestingly, those stable modifications are the only ones in vivo identified in the hemoglobin of moderate alcohol drinkers.